Method of allele-specific amplification

ABSTRACT

A method of selectively producing and amplifying a cDNA sequence of a target allele of a gene, wherein the target allele is a mutant allele or is a specific allele of a polymorphic gene, the method comprising: (a) providing a sample comprising an mRNA transcript of the target allele; (b) performing a reverse-transcription reaction to generate a cDNA sequence from the mRNA transcript, and (c) amplifying the cDNA of the target allele; wherein the reverse-transcription reaction is selective for reverse transcription of the mRNA transcript of the target allele over an mRNA transcript of an alternative allele of the same gene.

FIELD OF THE INVENTION

The present invention relates to a method of selectively producing andamplifying a cDNA sequence of a target allele of a gene. The presentinvention also relates to a kit for selectively producing and amplifyinga cDNA sequence of a target allele of a gene.

BACKGROUND OF THE INVENTION

Risk of disease and response to treatment varies from person to person.This is to due to variation in human genetic coding, interactionsbetween genes and the environment over a lifetime, and the uniquesignature of the immune system. Defining the scope and nature of humanbiological variation has allowed, and will continue to allow,assessments to be made regarding disease diagnosis, disease prognosisand the targeting of medical treatments to those that will most likelybenefit.

Variations in DNA between individuals can be caused by DNA mutations.Mutagenesis can lead to sudden and spontaneous changes in a cell and canarise from a number of different possible causes, including radiation,viruses, transposons and mutagenic chemicals, as well as errors thatoccur during cell division or DNA replication. They can also be inducedby the organism itself by cellular processes such as hypermutation.Mutations exist in different forms, such as point mutations, insertionsand deletions. Mutations such as point mutations that occur withinprotein coding regions of gene can lead to erroneous codon codes whichcan e.g. code for a different amino acid or code for a stop codonresulting in a truncated form of a protein. Mutations may also lead toframeshifts caused by the insertion or deletion of nucleotides,resulting in a completely different translation from the wild-typesequence. Mutations may lead to loss of function of a protein, gain offunction, or may act antagonistically to the wild-type allele.

Mutations can provide important genetic markers for disease diagnosis orprognosis. Furthermore, the identification of mutations can play asignificant role in helping to tailor drugs and drug regimens toparticular genotypes.

Mutational profiling of key cancer pathway genes is becoming commonpractice in the way therapies are being selected for patient care. Somealterations have been shown to increase sensitivity to a certain drugwhile other mutations result in decreased sensitivity or even resistanceto a given therapy. There are a number of reports in the literature thatdocument the increased sensitivity to erlotinib seen in NSCLC patientswith to an L858R and other EGFR mutations. Additionally, resistance toimatinib seen in patients with CML has been associated with mutations inthe kinase domain of the c-abl gene involved in the BCR-ABL fusion gene.Although identification of these mutations has become important forpatient care, these therapies were not originally developed to targetthe specific alterations. In more recent years specific alterationsidentified in subsets of patients are being used to develop targetedtherapies against the key alterations. Two recent examples of the trendtoward such targeted therapies are crizotinib and vemurafenib forpatients with EML4-ALK fusions and BRAF V600E mutations, respectively.Vemurafenib has been approved by the Food and Drug Administration totreat patients with metastatic melanoma who have a BRAF V600E mutation.The BRAF protein is normally involved in cell growth regulation, but ismutated in about half of patients with late-stage melanomas. Vemurafenibis a BRAF inhibitor that is able to block the function of theV600E-mutated BRAF protein from driving the proliferation of cancercells. As more therapies are developed against specific alterations theincreased need for sensitive and specific mutational profilingmethodologies is becoming more important.

Nucleic acid polymorphisms can also contribute to the genetic diversitybetween individuals. Polymorphisms can take several forms, includingsingle nucleotide substitutions, nucleotide insertions, and nucleotidedeletions. In the case of insertions and deletions, the insertion ordeletion of one or more nucleotides at a position in a gene may bepresent.

Single nucleotide polymorphisms (SNPs) represent an abundant form ofgenetic variation in humans. SNP patterns are likely to influence manyhuman phenotypes. Consequently, large scale association studies based onSNP genotyping are expected to help identify genes affecting complexdiseases and responses to drugs or environmental chemicals. SNPs canprovide important genetic markers for disease diagnosis or prognosis.Furthermore, the identification of SNPs (and other geneticpolymorphisms) can play a significant role in helping to tailor drugsand drug regimens to particular genotypes.

As a consequence of the clear impact that pharmacogentics can, and will,have on the healthcare industry, there is a pressing need to developimproved methods of genotype testing.

Various methods of allele discrimination methods are known in the art.Examples of such methods include allele specific hybridisation,allele-specific single-base primer extension, allele specific enzymaticcleavage, and allele-specific polymerase chain reaction (AS-PCR).

Allele specific hybridisation discriminates between alleles at a SNPlocus using allele-specific oligonucleotide probes. Stringencyconditions are employed such that a single-base mismatch is sufficientto prevent hybridisation of the non-matching probe. In allele-specificsingle-base extension, primers are designed that anneal one nucleotideupstream of the polymorphic or mutant site. In this method, allelediscrimination depends on the ability of this perfectly-annealed primerto be extended. Allele specific enzymatic cleavage employs fragmentlength polymorphism (RFLP) analysis. An RFLP is generated when amutation/SNP occurs at a restriction endonuclease recognition sequence,and one allele preserves the sequence while the other destroys it. Thepresence of a mutation/SNP can be detected from the number of cleavageproducts after application of a restriction enzyme.

Allele-specific polymerase chain reaction is a powerful method in whichallele discrimination is achieved by allele-specific primer annealing,followed by PCR amplification. Allele specific PCR is typicallyperformed on DNA samples using primers that have a complimentarynucleotide in the primer (e.g. in the 3′ position) in order toselectively amplify the intended target. Although this methodology workswell, it often requires a fair amount of optimisation and knowledgeabout primer/template interactions in order to obtain the requiredspecificity. The issue many times is that the discriminatory power ofthe single nucleotide change may not be sufficient to inhibitamplification of the wild-type allele to some degree. There are a numberof ways to manipulate the reaction and reagents reported in theliterature in order to attempt to increase specificity. However, thereare a number of different factors to consider other than just theaffinity of the primer for the intended template. One such factor is theamount of mutant target present in the background of wild-type alleles.Wild-type alleles that are present in the reaction mix make it much moredifficult to selectively amplify the intended mutant target as thewild-type alleles tend to bind primers and to some degree generatesignals that are indistinguishable from the intended amplification.

Renaud et al. (Journal of Clinical Virology; 49 (2010); 21-25) describea diagnostic assay employing an allele-specific reversetranscriptase-PCR (AS-RTPCR) assay that targets to the H275Y oseltamivirresistant mutation in 2009 pandemic influenza A.H1N1 virus. The methodemployed by Renaud uses a two-step RT-PCR reaction employing a commonreverse primer and probe and two-allele-specific forward primers(wild-type and mutant) which are designed to include the SNP of interestat the 3′ end. However, since the reverse primer (the primer for thereverse transcription step) was common for the two alleles, the reversetranscription reactions were not discriminatory for the transcripts ofthe mutant versus wild-type. Accordingly, any discriminating ability ofthe method required discrimination at the cDNA level.

Against this background, there is pressing need to develop improvedmethods of genotype testing, for example to distinguish between mutantand wild-type alleles. In particular, new methods are required thatimprove the sensitivity and specificity to enable accurate and efficientroutine testing procedures.

SUMMARY OF THE INVENTION

The present invention relates to a method of selectively producing andamplifying a target allele of a gene.

More particularly, the present invention provides a method ofselectively producing and amplifying a cDNA sequence of a target alleleof a gene, the method comprising:

-   a) providing a sample comprising an mRNA transcript of the target    allele;-   b) performing a reverse-transcription reaction to generate a cDNA    sequence from the mRNA transcript, and-   c) amplifying a cDNA sequence of the target allele generated in step    (b);    wherein the reverse-transcription reaction in step (b) is selective    for reverse transcription of the mRNA transcript of the target    allele over an mRNA transcript of an alternative allele of the same    gene.

The target allele may be a mutant allele or a specific allele of apolymorphic gene. Thus, the present invention provides a method ofselectively producing and amplifying a cDNA sequence of a target alleleof a gene, wherein the target allele is a mutant allele or is a specificallele of a polymorphic gene, the method comprising:

-   a) providing a sample comprising an mRNA transcript of the target    allele;-   b) performing a reverse-transcription reaction to generate a cDNA    sequence from the mRNA transcript, and-   c) amplifying a cDNA sequence of the target allele generated in step    (b);    wherein the reverse-transcription reaction in step (b) is selective    for reverse transcription of the mRNA transcript of the target    allele over an mRNA transcript of an alternative allele of the same    gene.

The present invention thus relates to a method that helps to eliminatethe presence of unwanted alleles (e.g. wild-type alleles) in the nucleicacid (e.g. cDNA) population. This is accomplished by selectively turningthe allele target (e.g. mutant allele) mRNA into potential targets foramplification, while leaving the unwanted allele (e.g. wild-type allele)in the form of RNA which is not a substrate for amplification. Thisconversion of (e.g. mutant) allele mRNA to cDNA happens in the initialreverse transcription step of the reaction. As the reaction proceeds toamplification by, for example a polymerase chain reaction (PCR), theonly targets present in the sample are the target (e.g. mutated) copiesof cDNA which helps to eliminate the potential for mis-priming.

The present invention thus allows for significant advantages overconventional allele specific PCR. Firstly, by only priming thetranscript of interest (e.g. mutant), the only cDNA that is generated isfrom the transcripts of interest (e.g. mutant transcripts), in effecteliminating the potential for mis-priming and reducing unwantedbackground signals. Secondly, the method increases the number of targetsper cell equivalence over the single copy of DNA that would be availableusing standard AS-PCR e.g. if the gene is transcribed at a rate of morethan one copy from the allele of interest (e.g. mutated allele) percell. This is particularly advantageous as the number of cells availablefor testing becomes limited, as is often the case with e.g. circulatingtumour cell (CTC) analysis and other small biopsy samples. The presentinventors have demonstrated this successfully using patient samplescomprising the V600E mutation. When performing a traditional allelespecific PCR, the reaction can generate a significant amount ofbackground in known wild-type samples. Additionally, the level ofsensitivity is in the region of 1-5%. However, when using the method ofthe present invention, the background signal is improved and asignificant gain in sensitivity is achieved. Thus, the method of thepresent invention can serve as a way to increase available targets fordetection of mutations in rare event populations as well as increasingthe sensitivity and specificity of routine allele-specific testing.

In addition to helping in the selection of therapies and thedetermination of diagnosis or prognosis based on a mutation or polymorph(e.g. SNP) profile, the present invention may also be clinicallysignificant in being able to determine the presence of an active genecarrying a mutation or polymorph by detecting the mutation or polymorphat the transcript level. This provides an additional level of confidencethat the mutated allele is actually driven from an active promoter andtherefore produces the targeted protein. Accordingly, the invention asdescribed herein may also serve as an attractive alternative in theabsence of robust antibodies to detect variant proteins.

Additionally, the invention described herein may provide valuableinformation about the quantity of a mutant transcript present in asample as a means to monitor drug efficacy and disease progression.Quantitative DNA based PCR for mutations associated with hematologicmalignancies such as abl T315I and JAK2 V617F are currently in usetoday. Although there is currently no targeted therapy for patients witha JAK2 V617F mutation, there are a number of development effortsunderway which target this mutation in patients with Polycythemia Vera.Monitoring the efficacy of a JAK2 inhibitor with a quantitative JAK2V617F determination at the transcript level may provide clinicallyrelevant information similar to how BCR-ABL RT-PCR is used formonitoring therapy and disease progression in patients with CML today.

In a preferred embodiment of the present invention, the target allele isa mutant allele. In a further preferred embodiment, the target allele isa mutant allele and the alternative allele is the wild-type allele. Themutant allele may be, for example, the result of a point mutation,nucleotide insertion(s) or a nucleotide deletion(s). In a preferredembodiment, the method is used to amplify a mutant allele comprising aspecific point mutation that is not present in the alternative allele.In a preferred embodiment, the mutant allele comprises a singlenucleotide substitution that is not present in the alternative allelei.e. the method can discriminate between a sequence containing thenucleotide substitution versus the corresponding sequence that does notcontain the nucleotide substitution.

The target allele may also be a specific allele of a polymorphic genecomprising a polymorphic site, the target allele and alternative allelediffering in base composition at the polymorphic site. The polymorphismmay be, for example, in the form of a single nucleotide polymorphism(SNP), nucleotide insertion(s) or nucleotide deletion(s). In a preferredembodiment, the polymorphic site is a SNP site.

In a preferred embodiment, the reverse-transcription reaction comprises(i) annealing a reverse primer to a region of the mRNA transcript of thetarget allele comprising a target site and (ii) extending the reverseprimer to generate a cDNA sequence from the mRNA transcript of thetarget allele, wherein the mRNA transcript of the target allele and themRNA of the alternative allele differ in base composition at theposition of the target site, and wherein selectivity for reversetranscription of the target allele mRNA over the alternative allele mRNAis achieved by the presence of one or more bases in the reverse primerwhich are complementary to the mRNA sequence at the target site of thetarget allele but which establish a mis-match at the position of thetarget site in the alternative allele. The target site may be, forexample, a mutation site or a polymorphic (e.g. SNP) site, as describedherein.

The reverse primer used in the present invention may bind the targetsite with full complementarity to the mRNA of the target allele or withone or more base mis-matches may be present. In a particularly preferredembodiment, the reverse primer binds with full complementarity (i.e. nobase-base mis-matches) to the mRNA of the target allele.

The selectivity for reverse transcription of the target allele mRNA overthe alternative allele mRNA can be achieved, at least in part, by a baseat the 3′ end of the reverse primer which establishes a mis-match withthe mRNA sequence of the alternative allele but which base-pairs withthe mRNA sequence of the target allele.

In a particularly preferred embodiment, the amplification step comprisesperforming a polymerase chain reaction (PCR) on the generated cDNAsequence of the target allele. In a preferred aspect of this embodiment,the reverse transcription reaction and PCR reaction employ the samereverse primer. The forward primer and reverse primer employed in thePCR reaction may each bind to a region of the target allele derived fromthe same exon and/or the reverse transcription reaction and PCR reactionare carried out using the same enzyme, optionally wherein the enzyme isrTth.

In an embodiment of the present invention, the target allele that isselectively amplified by the present invention may be an allele of HER2,PI3K (PIK3CA), KRAS, EGFR, c-MET, MEK (MEK1 or MEK2), PTEN, NRAS, HRAS,FGFR1, JAK2, ABL (also known as ABL1 and c-able oncogene 1, non-receptortyrosine kinase), BRAF or ALK. For example, the target allele may beselected from the group consisting of BRAF V600E, BRAF V600D, BRAFV600R, BRAF V600K, EGFR L858R, EGFR T790M, ALK C1156Y and ABL T315I. Inone embodiment, the targets recited herein may be part of gene fusionconstructs encoding fusion proteins (e.g. EML4-ALK fusions and BCR-ABLfusions).

In a particularly preferred embodiment of the present invention, theselectively amplified cDNA is detected and/or quantified e.g. byreal-time PCR.

In a further aspect of the invention, the presence of the target alleleis predictive of a diagnosis and/or a prognosis of a subject from whichthe sample is taken. In an embodiment of this aspect, the method maycomprise detecting and/or quantifying the amplified cDNA of the targetallele and assessing from the detection/quantitation of the amplifiedcDNA a diagnosis and/or a prognosis of the subject.

In a further aspect, the sample that is analysed in the presentinvention is from a subject known to have, or suspected to have, adisease, and the presence of the target allele is predictive of how thesubject will respond to administration of a drug to treat the disease.In an embodiment of this aspect, the method may comprise detectingand/or quantitating the amplified cDNA of the target allele andassessing from the detection/quantitation of the amplified cDNA thelikelihood of success of treating the subject with the drug. Forexample, the disease may be cancer, the target allele can be the mutantallele of the human BRAF gene encoding the V600E mutation and the drugcan be vemurafenib.

The present invention also provides a kit for selectively producing andamplifying a cDNA sequence of a target allele of a gene by reversetranscription PCR, wherein the target allele is a mutant allele or is aspecific allele of a polymorphic gene, and wherein the kit comprises:(i) a reverse primer specific to a region of an mRNA transcript of thetarget allele comprising a target site, wherein the mRNA transcript ofthe target allele of the gene and the mRNA of an alternative allele ofthe gene differ in base composition at the position of the target site,and wherein the reverse primer comprises one or more bases which arecomplementary to the mRNA sequence at the target site of the targetallele but which establish a mis-match at the position of the targetsite in the alternative allele; (ii) a forward primer specific for anupstream region of the target allele; (iii) a reverse transcriptase; and(iv) a DNA polymerase.

In a preferred embodiment, the reverse transcriptase and the DNApolymerase of the kit are the same enzyme.

In a further aspect, provided herein is a method of detecting for thepresence of a gene mutation, the method comprising:

-   a) providing a sample comprising an mRNA transcript;-   b) contacting the sample with reagents capable of performing a    reverse-transcription reaction when mRNA containing the mutation is    present, thereby generating a cDNA sequence from the mRNA transcript    when mRNA containing the mutation is present; and-   c) amplifying, if present, any of the said cDNA sequence generated    in step (b);    wherein the reverse-transcription reaction in step (b) is selective    for reverse transcription when the mRNA transcript containing the    mutation is present over the alternative transcript of the gene that    does not contain the mutation.

The reagents in step (b) of this aspect will typically comprise areverse primer which is selective for reverse transcription of the mRNAcontaining the mutation by the presence of a base in the reverse primerwhich is complementary to the mRNA base containing the mutation butwhich establishes a mismatch in the alternative transcript. The base ispreferably at the 3′ end of the reverse primer. Step (c) preferablycomprises annealing a forward primer to the cDNA sequence and performinga polymerase chain reaction (PCR) on the cDNA sequence. The reversetranscription reaction and PCR reaction may employ the same reverseprimer. The mutation may be any of the mutations described above, andmay be for example mutations in HER2, PI3K (PIK3CA), KRAS, EGFR, c-MET,MEK, PTEN, NRAS, HRAS, FGFR1, JAK2, ABL, BRAF or ALK.

In a further aspect, the present invention provides a method ofdetecting for the absence of a gene mutation, the method comprising:

-   a) providing a sample comprising an mRNA transcript from the gene;-   b) performing a reverse-transcription reaction to generate a cDNA    sequence from the mRNA transcript; and-   c) amplifying the cDNA sequence generated in step (b);    wherein the reverse-transcription reaction in step (b) is selective    for reverse transcription of the mRNA transcript when the mutation    is absent over the corresponding mRNA transcript containing the    mutation.

In this aspect, the reverse transcription reaction may comprise:

-   (i) annealing a reverse primer to a region of the mRNA containing    the mutation site; and-   (ii) extending the reverse primer to generate a cDNA sequence from    the mRNA transcript; wherein selectivity for reverse transcription    of the mRNA is achieved, at least in part, by the presence of a base    which establishes a mismatch in the mRNA transcript having the    mutation at the mutation site. The base is preferably at the 3′ end    of the reverse primer. Step (c) preferably comprises annealing a    forward primer to the cDNA sequence and performing a polymerase    chain reaction (PCR) on the cDNA sequence. The reverse transcription    reaction and PCR reaction may employ the same reverse primer. The    mutation may be any of the mutations described above, and may be for    example mutations in HER2, PI3K (PIK3CA), KRAS, EGFR, c-MET, MEK,    PTEN, NRAS, HRAS, FGFR1, JAK2, ABL, BRAF or ALK.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a and FIG. 1 b show amplification plots generated from sample 1using DNA (FIG. 1 a) and mRNA (FIG. 1 b) as the starting material. FIG.1 a has a Ct value of 31.8 and FIG. 1 b has a Ct value of 25.8.

FIGS. 2 a and 2 b show amplification plots generated from sample 2 usingDNA (FIG. 2 a) and mRNA (FIG. 2 b) as the starting material. FIG. 2 ahas a Ct value of 24.4 and FIG. 2 b has a Ct value of 23.6.

FIGS. 3 a and 3 b show amplification plots generated from sample 3 usingDNA (FIG. 3 a) and mRNA (FIG. 3 b) as the starting material. FIG. 3 ahas a Ct value of 33.2 and FIG. 3 b has a Ct value of 25.7.

FIGS. 4 a and 4 b show amplification plots generated from sample 4 usingDNA (FIG. 4 a) and mRNA (FIG. 4 b) as the starting material. FIG. 4 ahas a Ct value of 25.4 and FIG. 4 b has a Ct value of 25.3.

FIGS. 5 a and 5 b show amplification plots generated from sample 5 usingDNA (FIG. 5 a) and mRNA (FIG. 5 b) as the starting material. FIG. 5 ahas a Ct value of 34 and FIG. 5 b has a Ct value of 26.3.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions apply to the present invention:

The term “allele” refers to a particular form of a genetic locus,distinguished from other forms by its particular nucleotide or aminoacid sequence.

The term “target allele” refers to an allele that is to be selectivelyamplified using the method of the present invention. The target allelemay represent a mutant or polymorphic variant that is present in apopulation at lower frequency. The target may also be a wild-typeallele. In some cases, two or more alleles of a given gene may have thesame mutation or polymorphism in common that is to be detected by themethod of the present invention. In such cases, a target allele maycomprise two or more alleles that share a mutation of interest.

The term “alternative allele” refers to an alternative allele of thetarget allele gene. The alternative allele will code for an mRNAsequence that differs from the mRNA sequence coded by the target allele.The method of the present invention is capable of selectively producingand amplifying cDNA of the target allele over producing and amplifyingcDNA the alternative allele when the target allele and alternativeallele are in the same sample. The mRNA transcript sequence from thetarget allele and the mRNA transcript sequence from the alternativeallele may, for example, differ only in the base or bases present (orabsent) at a single mutant site or polymorphic site.

The term “gene” refers to a hereditary unit consisting of a sequence ofDNA that occupies a specific location on a chromosome and determines aparticular characteristic in an organism.

The term “locus” refers to a location on a chromosome or DNA moleculecorresponding to a gene or a physical or phenotypic feature.

The term “nucleic acid” refers to a single stranded or double strandedDNA or RNA molecule including natural nucleic acids found in natureand/or modified, artificial nucleic acids having modified backbones orbases, as are known in the art.

The term “polymorphic site” refers to a position within a locus at whichat least two alternative bases or sequences are found in a population.

The term “polymorphism” refers to the sequence variation observed in anindividual at a polymorphic site. Polymorphisms include nucleotidesubstitutions, insertions, deletions, and may, but need not, result indetectable differences in gene expression or protein function.

The term “primer” refers to a molecule that physically hybridizes with atarget nucleic acid. The primer is capable of being extended in anamplification reaction such as a PCR reaction or in areverse-transcription reaction. Typically, a primer can be made from, orcomprise of, any combination of nucleotides or nucleotide derivatives oranalogs available in the art. More typically, a primer will be in theform of an oligonucleotide. Primers may also contain one or morenucleotide alternatives or modified bases to add increased specificityand/or disrupt the efficiency of primer extension in the presence of amis-match. Alternative bases used to enhance specificity may includeLocked Nucleic Acid (LNA) bases, Peptide Nucleic Acid (PNA) bases andInosine. The primer may be unlabelled or labelled with a detectionmarker.

The present invention provides a method of selectively producing andamplifying a cDNA sequence of a target allele of a gene, wherein thetarget allele is a mutant allele or is a specific allele of apolymorphic gene, the method comprising:

-   a) providing a sample comprising an mRNA transcript of the target    allele;-   b) performing a reverse-transcription reaction to generate a cDNA    sequence from the mRNA transcript, and-   c) amplifying a cDNA sequence of the target allele generated in step    (b);    wherein the reverse-transcription reaction in step (b) is selective    for reverse transcription of the mRNA transcript of the target    allele over an mRNA transcript of an alternative allele of the same    gene. Accordingly, when the target allele of the gene and the    alternative allele of the gene are present in the same sample, the    method of the present invention is able to selectively produce and    amplify a cDNA sequence of a target allele of a gene over producing    and amplifying a cDNA sequence of the alternative allele. In one    embodiment, the target allele and the alternative allele DNA and/or    RNA are both present in the sample. However, it will be appreciated    in some cases the method of the present invention can be carried out    on a sample where it is not known whether both the target allele and    alternative allele nucleic acid are present in the sample e.g. the    sample may contain target allele but not the alternative allele (or    vice-versa). The method of the present invention is also    advantageous in such cases since detection of amplification product    resulting from the method of the present invention can be used to    confirm the presence of the target allele.

For example, the method of the present invention may be used to detectfor the presence of a gene mutation, the method comprising:

-   a) providing a sample comprising an mRNA transcript;-   b) contacting the sample with reagents capable of performing a    reverse-transcription reaction when mRNA containing the mutation is    present, thereby generating a cDNA sequence from the mRNA transcript    when mRNA containing the mutation is present; and-   c) amplifying, if present, any of the said cDNA sequence generated    in step (b);    wherein the reverse-transcription reaction in step (b) is selective    for reverse transcription when the mRNA transcript containing the    mutation is present over the alternative transcript of the gene that    does not contain the mutation.

The method may also be used to detect for the absence of a genemutation, the method comprising:

-   a) providing a sample comprising an mRNA transcript from the gene;-   b) performing a reverse-transcription reaction to generate a cDNA    sequence from the mRNA transcript; and-   c) amplifying the cDNA sequence generated in step (b);    wherein the reverse-transcription reaction in step (b) is selective    for reverse transcription of the mRNA transcript when the mutation    is absent over the corresponding mRNA transcript containing the    mutation.

In the reverse transcription reaction, a DNA molecule (termed acomplementary DNA molecule, which can be abbreviated to a “cDNAmolecule”) is generated from a single stranded RNA template through theenzyme reverse transcriptase. Generating cDNA from mRNA is well known inthe art. The cDNA sequence may be generated from the full length of themRNA sequence or a portion of the mRNA sequence. A skilled person canreadily determine the appropriate annealing and extension temperaturesfrom the primer sequence, mRNA template and choice of reversetranscriptase using procedures well known in the art. In one embodiment,reverse transcription is executed by rTth.

The selectivity of the method can be achieved, for example, by annealinga reverse primer to a region of the mRNA transcript of the target allelethat contains the only difference in sequence between the mRNAtranscript of the target allele and the mRNA transcript of thealternative allele.

In a preferred embodiment, the selectivity of the method can be achievedby annealing a reverse primer to a region of the mRNA transcript of thetarget allele comprising a target site and extending the reverse primerto generate a cDNA sequence from the mRNA transcript of the targetallele, wherein the mRNA transcript of the target allele and the mRNA ofthe alternative allele differ in base composition at the position of thetarget site. In this way, the selectivity for reverse transcription ofthe target allele mRNA over the alternative allele mRNA is achieved bythe presence of one or more bases in the reverse primer which arecomplementary to the mRNA sequence at the target site when the primer ishybridized to the mRNA transcript of the target allele but whichestablishes a mismatch at the position of the target site in thealternative allele. A mismatch may be established, for example, by thedisruption or removal of one or more non-covalent bonds, such as one ormore hydrogen bonds e.g. by disrupting or removing a Watson-Crick basepair.

A person skilled in the art would be able to generate allele-specificprimers using methods known in the art. In a preferred embodiment, andas exemplified in the specific examples disclosed herein, the reverseprimer is designed to have a residue at the 3′ terminus of the primerthat is complimentary to the mRNA of the target allele and not to mRNAof another (or the other) alternative allele of the gene. Thus, thereverse primer may be designed to have a base at the 3′ terminus of theprimer that, when the primer is annealed to the mRNA of the targetallele, is complementary to a base present at the target site (e.g.mutant site or polymorphic site) of the target allele but is notcomplementary to a base present in the corresponding position of thealternative allele. Modifications to the primer adjacent to the 3′terminal base may also create enough disruption of the primer bindingefficiency to effectively disable primer extension. Additionally,modified bases known to increase hybridization stringency such as LNAand PNA bases may also be substituted at strategic positions of theprimer. The presence of the “mismatch” (e.g. by removing or disruptingthe formation of a base-pair) at the 3′ end of the reverse primerdisrupts the ability of the reverse transcriptase to extend the primer.A skilled person would readily be able to confirm whether a reverseprimer has the desired selectivity by performing a reverse transcriptionreaction in the presence of target allele and alternative allele anddetecting the level of cDNA production.

In a preferred embodiment, the alternative allele is complementary alongthe full length of the reverse primer with the exception of a singlenon-complementary mis-match between the base at the 3′ end of thereverse primer and the corresponding base of the alternative allele.However, the alternative allele may be complementary along the fullsequence of the reverse primer with the exception of two or morebase-pair mis-matches (e.g. 2, 3, 4, 5 or more) between the reverseprimer and the corresponding base of the alternative allele.

In a preferred embodiment of the present invention, the reverse primerhybridizes to an mRNA transcript of the target allele with fullcomplementarity to the mRNA of the target allele. By this is meant thateach base of the reverse primer forms a base-pair with a complementarybase on mRNA transcript when the primer is hybridized to the mRNAtranscript.

The reverse primer of the present invention can be a nucleic acidsequence, preferably a DNA oligonucleotide. The primer is of sufficientlength to enable reverse transcription of an mRNA transcript of thetarget allele. The primer may be, for example, in the range of 10-50nucleotides in length, preferably about 10-35 nucleotides, morepreferably about 10-30 nucleotides in length.

In the method of the present invention, the cDNA molecule that isgenerated from the mRNA transcript of the target allele is subjected toan amplification reaction. It should be noted that references throughoutthis disclosure to amplifying a cDNA sequence of the target allelegenerated in step (b) encompasses amplification of either the completecDNA sequence generated in step (b) or a part of the cDNA sequencegenerated in step (b). Amplification of DNA is an established procedurein molecular biology and can be carried out by many alternative methodsknown in the art. Example amplification methods include thermal cyclerbased amplification with thermostable enzymes (e.g. polymerase chainreaction (PCR), ligase chain reaction (LCR), in-situ amplification, longdistance PCR, digital PCR, real-time PCR, multiplex PCR andligation-dependent probe amplification), and isothermal amplification(e.g. strand displacement amplification (SDA), real-time stranddisplacement amplification, loop mediated isothermal amplification,ligation mediated rolling circle amplification, rolling circleamplification, and multiple displacement amplification).

Descriptions of amplification techniques can be found in, among otherplaces, Sambrook and Russell; Sambrook et al.; Ausbel et al.; PCRPrimer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press(1995); The Electronic Protocol Book, Chang Bioscience (2002) (“TheElectronic Protocol Book”); Msuih et al., J. Clin. Micro. 34:501-07(1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., HumanaPress, Totowa, N.J. (2002)(“Rapley”); U.S. Pat. No. 6,027,998; Barany etal., PCT Publication No. WO 97/31256; Wenz et al., PCT Publication No.WO 01/92579; Ehrlich et al., Science 252:1643-50 (1991); Innis et al.,PCR Protocols: A Guide to Methods and Applications, Academic Press(1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenauet al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin,Development of a Multiplex Ligation Detection Reaction DNA Typing Assay,Sixth International Symposium on Human Identification, 1995 (availableon the world wide web at: promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR Kit Instruction Manual, Cat.#200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci.USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951(1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al.,Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene109:1-11 (1991); Walker et al., Nucl. Acid Res. 20:1691-96 (1992);Polstra et al., BMC Inf. Dis. 2:18-(2002); and Landegren et al., Science241:1077-80 (1988).

In a particularly preferred embodiment, the amplification methodemployed in the present invention is a PCR-based amplification method.Polymerase chain reaction (PCR) is very widely known in the art. Forexample, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; K. Mullis,Cold Spring Harbor Symp. Quant. Biol., 51:263-273 (1986); and C. R.Newton & A. Graham, Introduction to Biotechniques: PCR, 2.sup.nd Ed.,Springer-Verlag (New York: 1997), the disclosures of which areincorporated herein by reference, describe processes to amplify anucleic acid sample target using PCR amplification extension primerswhich hybridize with the sample target.

Using PCR, the cDNA is amplified exponentially using a polymerase e.g. aDNA polymerase. PCR requires forward and reverse extension primers whichhybridize with the sample target. As the PCR amplification primers areextended, using a DNA polymerase (preferably thermostable), more sampletarget is made so that more primers can be used to repeat the process,thus amplifying the sample target sequence. Typically, the reactionconditions are cycled between those conducive to hybridization andnucleic acid polymerization, and those that result in the denaturationof duplex molecules. To briefly summarize, in the first step of thereaction, the nucleic acid molecules of a sample are transiently heated,in order to denature double stranded molecules. Forward and reverseprimers are present in the amplification reaction mixture at an excessconcentration relative to the sample target. When the sample is cooledto a temperature conducive to hybridization and polymerization, theprimers hybridize to the complementary sequence of the nucleic acidmolecule at a position 3′ to the sequence of the region desired to beamplified that is the complement of the sequence whose amplification isdesired. Upon hybridization, the 3′ ends of the primers are extended bythe polymerase. The extension of the primer results in the synthesis ofa DNA molecule having the exact sequence of the complement of thedesired nucleic acid sample target. The PCR reaction is capable ofexponentially amplifying the desired nucleic acid sequences, with a neardoubling of the number of molecules having the desired sequence in eachcycle. Thus, by permitting cycles of denaturation, hybridization, andpolymerization, an exponential increase in the concentration of thedesired nucleic acid molecule can be achieved. A preferred physicalmeans for strand separation involves heating the nucleic acid until itis completely (>99%) denatured. Typical heat denaturation involvestemperatures ranging from about 80° C. to about 105° C., for timesranging from a few seconds to minutes.

In the present invention, the template for amplification is the cDNAstrand produced during the reverse transcription step. Accordingly, thePCR reaction would typically require a forward primer that anneals tothe cDNA strand produced during the reverse transcription step and whichis then extended using an enzyme with DNA polymerase activity to producethe complement cDNA strand. The resulting cDNA strand can then bedenatured and the forward primer and a reverse primer annealed to therespective cDNA strands to allow further extension. The primers are thenextended by the polymerase to replicate the cDNA sequences, and theprocess is then repeated multiple times.

In a particularly preferred embodiment, the reverse primer used for thereverse transcription step is also used as the reverse primer for thePCR amplification step. This is advantageous in that the reversetranscription step and the PCR amplification can be carried out using asingle cocktail of reagents, thereby allowing a “one-step” reaction. Thecocktail of reagents may comprise a reverse transcriptase enzyme for thereverse transcription step and a DNA polymerase enzyme for the PCRreaction. However, in a preferred aspect of the invention, a singleenzyme is used that is able to perform the enzymatic steps in both thereverse transcription reaction and the PCR reaction. An example of suchan enzyme that is configured for DNA polymerization and reversetranscription that can be used in the present invention is rTth.

In a further embodiment, the forward primer and reverse primer arespecific for the same exon.

In an alternative embodiment, for example where contaminating DNA ispresent, primer pairs may be employed that span intron-exon boundariesto further prevent genomic DNA from being amplified. For example, theforward primer may hybridize to a region of the cDNA derived from twodifferent exons. Alternatively, each member of a primer pair may spandifferent exons.

The term “sample” is used in a broad sense herein and is intended toinclude a wide range of biological materials as well as compositionsderived or extracted from such biological materials. Exemplary suchmaterials or samples include whole blood; red blood cells; white bloodcells; buffy coat; hair; nails and cuticle material; swabs, includingbut not limited to buccal swabs, throat swabs, vaginal swabs, urethralswabs, cervical swabs, throat swabs, rectal swabs, lesion swabs, abcessswabs, nasopharyngeal swabs, and the like; urine; sputum; circulatingtumour cells (CTCs); exosomes; microsomes; cell free nucleic acid;saliva; semen; lymphatic fluid; amniotic fluid; cerebrospinal fluid;peritoneal effusions; pleural effusions; fluid from cysts; synovialfluid; vitreous humor; aqueous humor; bursa fluid; eye washes; eyeaspirates; plasma; serum; pulmonary lavage; lung aspirates; and tissues,including but not limited to, liver, spleen, kidney, lung, intestine,brain, heart, muscle, pancreas, biopsy material, and the like. Theskilled artisan will appreciate that lysates, extracts, or materialobtained from any of the above exemplary biological samples are alsowithin the scope of the invention. Tissue culture cells, includingexplanted material, primary cells, secondary cell lines, and the like,as well as lysates, extracts, or materials obtained from any cells, arealso within the meaning of the term biological sample as used herein. Inone embodiment the sample is derived from a tissue section. Tissuesections may be formalin-fixed paraffin embedded tissue sections. Suchsections may be incubated in digestion buffer to release the cellularcontent.

The samples used in the practice of the present invention may beobtained or derived from any source that contains, or is considered topotentially contain an mRNA transcript of a target allele or a part ofsaid mRNA transcript. The mRNA-containing sample may, for example, beobtained or derived from any mammal. Preferably the mRNA-containingsample is obtained from or derived from a human. In one embodiment, themRNA is obtained or derived from a subject known to have or suspected ofhaving a disease. An example of such a disease is cancer. The cancer maybe prostate, breast, lung, ovarian, pancreatic, bowel, colon, stomach,skin cancer, metastatic melanoma, or a brain tumour or malignancyaffecting the bone marrow (including the leukaemias) andlymphoproliferative systems, such as Hodgkin's or non-Hodgkin'slymphoma.

In a preferred embodiment of the present invention, the sample to beused in the method of the present invention can be pre-processed, forexample to remove contaminating DNA and/or purify RNA (including mRNA)in the sample. Methods for DNA removal are known in the art, such asacid phenol:chloroform extraction or Lithium chloride precipitation, andcan be followed DNase digestion. In one embodiment the DNA and RNAs maybe separated using column-based extraction protocols. Standardmethodologies for extracting nucleic acid in a test sample are wellknown in the art (see, for example, Sambrook et al. “Molecular Cloning—ALaboratory manual”, second edition. Cold Spring Harbor, N.Y. (1989)). Ina particularly preferred embodiment, the sample contains RNA that hasbeen isolated from contaminating DNA. Isolation of RNA is a routineprocedure in the art and there are multiple commercially available kitsfor this purpose (e.g. Strategene RNA Isolation kit).

The method of the present invention may further comprise detecting theamplified cDNA generated by reverse transcription of an mRNA transcriptof the target allele. The method of detection may vary depending on themethod used for the amplification step.

Many methods of detecting DNA sequences are known in the art. Thesimplest method of detection of nucleic acid amplification products isagarose gel electrophoresis. Products are separated based on mass byelectrophoresis through an agarose gel. The gels are then stained withethidium bromide, or an alternative such as SYBR green, which causenucleic acids to fluoresce under UV light. Stains used in agarose gelelectrophoresis give off a fluorescent signal when intercalated intoDNA, but not when unincorporated. Positive results are those in which aband of the appropriate size is present, while negative results lack theappropriate band.

A preferred method of detecting the amplified cDNA in the presentinvention is employing real-time PCR. Real-time PCR allows amplificationof the target nucleic acid to be visualized in real time. Real-timedetection can be accomplished in a number of alternative ways. A firstway is through the use of a DNA intercalating fluorescent dye. This typeof reaction uses two primers just like a standard PCR, but also requiresthe addition of an intercalating dye. An example of such a dye is SYBRgreen. In this type of assay, as the specific target amplifies, more dyebecomes incorporated into DNA and the fluorescent signal increases. Asecond type of real-time PCR detection also uses two primers, butemploys a fluorescently labeled oligonucleotide probe. This secondmethod works on the principle that a fluorescent dye (the reporter) isattached to one end of the oligonucleotide and a quencher, which absorbslight emitted from the reporter when in close proximity to it, is boundto the other end. The close proximity of the reporter to the quencherprevents detection of its fluorescence; breakdown of the probe by the 5′to 3′ exonuclease activity of the amplification polymerase breaks thereporter-quencher proximity and thus allows unquenched emission offluorescence, which can be detected after excitation with a lightsource. An increase in the product targeted by the reporter probe ateach PCR cycle therefore causes a proportional increase in fluorescencedue to the breakdown of the probe and release of the reporter. Anadditional advantage of real-time PCR is its quantitative nature. Eachpositive sample is given a Ct (defined as the PCR cycle in which thelevel of fluorescence has crossed the threshold). By running a series ofsamples containing known quantities of target RNA or DNA, a standardcurve can be created that correlates sample Ct to the initial quantityof target RNA or DNA in a sample. Unknown samples are then tested andtheir Ct values are compared with the standard curve to determine theinitial quantity of target RNA/DNA present in the sample. Alternatively,relative quantification can be assessed based on internal referencegenes to determine fold-differences in expression of the target gene.

Amplified nucleic acid may also be detected by labelling one of theprimers (e.g. forward or reverse) primer used in the amplificationprocess. Many methods for the detection of allelic variation aredescribed in standard textbooks, for example “Laboratory Protocols forMutation Detection”, Ed. by U. Landegren, Oxford University Press, 1996and “PCR”, 2^(nd) Edition by Newton & Graham, BIOS Scientific PublishersLimited, 1997.

By detecting the presence, or quantitating the amount of, target allele,the present invention can be used to draw conclusions regarding thesubject from which the test sample is taken. For example, target allelethat is detected in the present invention may be an allele that ispredictive of a diagnosis and/or a prognosis of a subject from which thesample is taken. Accordingly, by detecting and/or quantifying theamplified cDNA of the target allele, an assessment can be made as towhether the subject is likely to have a particular disease.Alternatively, by detecting and/or quantifying the amplified cDNA of thetarget allele, an assessment of the likely prognosis of the subject canbe made. An example of such a prognosis is the prediction of thelikelihood of a disease recurrence. An example of such a disease iscancer.

In a further embodiment of the present invention, the target allele thatis detected may be correlated with the likelihood of success of aparticular drug treatment. Great inroads have been made in the area ofpersonalised medicines and there are now multiple drug treatments thatare specific sub-populations that have a common genetic trait such as aspecific mutant allele. Accordingly, the present invention can be usedto detect for the presence of or quantify the amount of target allele ina sample derived from a subject, and the detection of, or amount of,target allele expression can be used in determining the treatment of thesubject with a drug associated with modifying (e.g. inhibiting) theexpression of the target allele or the activity of the protein expressedby the target allele.

An example is the treatment of erlotinib seen in non-small cell lungcancer (NSCLC) patients with EGFR mutations. One such mutation is EGFRL858R. Accordingly, in a further embodiment, the method of the presentinvention may be used to analyze a patient sample for expression (orabsence of expression) of a EGFR mutant allele or to quantify the levelof expression of EGFR mutant allele in a patient sample. In a furtheraspect of this embodiment, the patient has, or is suspected of havingNSCLC. In a yet further aspect, the present invention can be used todetect for the expression of a EGFR mutant allele in a sample derivedfrom a patient and the detection of, or level of EGFR mutant allele canbe used to decide, or aid, in the decision as to whether to administererlotinib or a drug capable of modifying the activity or expression ofEGFR. The EGFR mutant allele may be a L858 mutant allele (i.e. where theL residue at position 858 is replaced by a different (mutant) residue),preferably the EGFR L858R mutant allele.

Two recent examples of the trend towards targeted therapies arecrizotinib and vemurafenib for patients with EML4-ALK fusions and BRAFV600 mutations, respectively.

Vemurafenib which has been approved by the Food and Drug Administrationto treat patients with metastatic melanoma who have a BRAF V600mutation, such asV600E, V600K, V600D or V600R mutation. The BRAF proteinis normally involved in regulatory cell growth, but is mutated in abouthalf of patients with late-stage melanomas. Vemurafenib is a BRAFinhibitor that is able to block the function of the V600E-mutated BRAFprotein from driving the proliferation of cancer cells.

Accordingly, in one embodiment, the method of the present invention maybe used to analyze a patient sample for expression of a BRAF mutantallele or to quantify the level of expression of BRAF mutant allele. Ina further aspect of this embodiment, the to patient has, or is suspectedof having metastatic melanoma. In a yet further aspect, the presentinvention can be used to detect for the expression of a BRAF mutantallele in a sample derived from a patient and the detection of, or levelof BRAF mutant allele can be used to decide, or aid, in the decision asto whether to administer a specific therapy or a drug capable ofmodifying the activity or expression of BRAF. Preferably, the BRAFmutant allele is a V600 mutant allele (i.e. where the V residue atposition 600 is replaced by a different (mutant) residue). V600 mutantalleles may include V600E, V600K, V600D or V600R allele, more preferablythe V600E or V600K allele.

The protein sequence encoded by the human BRAF gene is set out below(SEQ ID NO: 10):

(SEQ ID NO: 10)              MAALSGGGGGGAEPGQALFNGDMEPEAGAGAGAAASSAADPAIPEEVWNIKQMIKLTQEHIEALLDKFGGEHNPPSIYLEAYEEYTSKLDALQQREQQLLESLGNGTDFSVSSSASMDTVTSSSSSSLSVLPSSLSVFQNPTDVARSNPKSPQKPIVRVFLPNKQRTVVPARCGVTVRDSLKKALMMRGLIPECCAVYRIQDGEKKPIGWDTDISWLTGEELHVEVLENVPLTTHNFVRKTFFTLAFCDFCRKLLFQGFRCQTCGYKFHQRCSTEVPLMCVNYDQLDLLFVSKFFEHHPIPQEEASLAETALTSGSSPSAPASDSIGPQILTSPSPSKSIPIPQPFRPADEDHRNQFGQRDRSSSAPNVHINTIEPVNIDDLIRDQGFRGDGGSTTGLSATPPASLPGSLTNVKALQKSPGPQRERKSSSSSEDRNRMKTLGRRDSSDDWEIPDGQITVGQRIGSGSFGTVYKGKWHGDVAVKMLNVTAPTPQQLQAFKNEVGVLRKTRHVNILLFMGYSTKPQLAIVTQWCEGSSLYHHLHIIETKFEMIKLIDIARQTAQGMDYLHAKSIIHRDLKSNNIFLHEDLTVKIGDFGLATVKSRWSGSHQFEQLSGSILWMAPEVIRMQDKNPYSFQSDVYAFGIVLYELMTGQLPYSNINNRDQIIFMVGRGYLSPDLSKVRSNCPKAMKRLMAECLKKKRDERPLFPQILASIELLAEDFSLYACASPKTPIQAGGYGAFPVH

In a further embodiment of the present invention, the target allele thatis detected may be correlated with resistance to particular drugtreatment. For example crizotinib can be used for patients with EML4-ALKfusions, particularly patients with NSCLC, anaplastic large celllymphoma, neuroblastoma, or other advanced tumors. It has beenidentified that the ALK C1156Y mutation in the tyrosine kinase domainconfers resistance to crizotinib. Accordingly, the method of the presentinvention may be used to analyze a patient sample for the expression ofan ALK mutant allele or to quantify the level of expression of an ALKmutant allele. In a further aspect of this embodiment, the patientshave, or are suspected of having NSCLC, anaplastic large cell lymphoma,or neuroblastoma, or other advanced tumors. In a yet further aspect, thepresent invention can be used to detect for the expression of an ALKmutant allele in a sample derived from a patient and the detection of,or level of ALK mutant allele can be used to decide, or aid, in thedecision as to whether to administer crizotinib or a drug capable ofmodifying the activity or expression of anaplastic lymphoma kinase(ALK). The ALK mutant allele may be an ALK C1156 mutant allele (i.e.where the C residue at position 1156 is replaced by a different (mutant)residue), preferably the ALK C1156Y allele.

A further example is the resistance to EGFR tyrosine kinase inhibitors(EGFR-TKIs) seen in patients with lung cancer. A secondary pointmutation that substitutes methionine in place of threonine at amino acidposition 790 (T790M) is a molecular mechanism that produces adrug-resistant variant of the targeted kinase. Accordingly, in a furtherembodiment, the method of the present invention may be used to analyze apatient sample for expression of an EGFR mutant allele or to quantifythe level of expression of an EGFR mutant allele. In a further aspect ofthis embodiment, the patient has, or is suspected of having lung cancer.In a yet further aspect, the present invention can be used to detect forthe expression of an EGFR mutant allele in a sample derived from apatient and the detection of, or level of EGFR mutant allele can be usedto decide, or aid, in the decision as to whether to administer an EGFRtyrosine kinase inhibitor. The EGFR mutant allele may be an EGFR T790mutant allele (i.e. where the T residue at position 790 is replaced by adifferent (mutant) residue), preferably the EGFR T790M allele.

Another example is the resistance to BCR-ABL inhibitors (e.g. imatinib)seen in patients with a T315I mutation in the ABL gene. It can be causedby a single cytosine to thymine (C->T) base pair substitution atposition 944 of the Abl gene (codon ‘315’ of the Abl protein) sequenceresulting in amino acid (T)hreonine being substituted by (I)soleucine atthat position—thus ‘T315I’.

Accordingly, in a further embodiment, the method of the presentinvention may be used to analyze a patient sample for expression of anABL mutant allele or to quantify the level of expression of an ABLmutant allele. In a further aspect of this embodiment, the patientshave, or are suspected of having chronic myelogenous leukemia. In a yetfurther aspect, the present invention can be used to detect for theexpression of an ABL mutant allele in a sample derived from a patientand the detection of, or level of ABL mutant allele can be used todecide, or aid, in the decision as to whether to administer imatinib ora BCR-ABL inhibitor. The ABL mutant allele may be a T315 mutant allele(i.e. where the T residue at position 315 is replaced by a different(mutant) residue), preferably the ABL T315I mutant allele.

The ABL protein sequence (also known as ABL1) is shown below (SEQ ID NO:11), with amino acid at position 315 highlighted:

(SEQ ID NO: 11)               MLEICLKLVGCKSKKGLSSSSSCYLEEALQRPVASDFEPQGLSEAARWNSKENLLAGPSENDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEWCEAQTKNGQGWVPSNYITPVNSLEKHSWYHGPVSRNAAEYLLSSGINGSFLVRESESSPGQRSISLRYEGRVYHYRINTASDGKLYVSSESRFNTLAELVHHHSTVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYII T EFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSAMEYLEKKNFIHRDLAARNCLVGENHLVKVADEGLSRLMTGDTYTAHAGAKFPIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDLSQVYELLEKDYRMERPEGCPEKVYELMRACWQWNPSDRPSFAEIHQAFETMFQESSISDEVEKELGKQGVRGAVSTLLQAPELPTKTRTSRRAAEHRDTTDVPEMPHSKGQGESDPLDHEPAVSPLLPRKERGPPEGGLNEDERLLPKDKKTNLFSALIKKKKKTAPTPPKRSSSFREMDGQPERRGAGEEEGRDISNGALAFTPLDTADPAKSPKPSNGAGVPNGALRESGGSGFRSPHLWKKSSTLTSSRLATGEEEGGGSSSKRFLRSCSASCVPHGAKDTEWRSVTLPRDLQSTGRQFDSSTFGGHKSEKPALPRKRAGENRSDQVTRGTVTPPPRLVKKNEEAADEVFKDIMESSPGSSPPNLTPKPLRRQVTVAPASGLPHKEEAGKGSALGTPAAAEPVTPTSKAGSGAPGGTSKGPAEESRVRRHKHSSESPGRDKGKLSRLKPAPPPPPAASAGKAGGKPSQSPSQEAAGEAVLGAKTKATSLVDAVNSDAAKPSQPGEGLKKPVLPATPKPQSAKPSGTPISPAPVPSTLPSASSALAGDQPSSTAFIPLISTRVSLRKTRQPPERIASGAITKGVVLDSTEALCLAISRNSEQMASHSAVLEAGKNLYTFCVSYVDSIQQMRNKFAFREAINKLENNLRELQICPATAGSGPAATQDFSKLLSSVKEISDIVQR

The corresponding ABL mRNA transcript (shown here in cDNA format wherebase u is replaced by t) is set out below (SEQ ID NO:12), with themutation base at position 947 highlighted in bold (where replacement ofc by t (u in the case of mRNA) gives rise to the T315I mutation).

(SEQ ID NO: 12)aaaatgttggagatctgcctgaagctggtgggctgcaaatccaagaaggggctgtcctcgtcctccagctgttatctggaagaagcccttcagcggccagtagcatctgactttgagcctcagggtctgagtgaagccgctcgttggaactccaaggaaaaccttctcgctggacccagtgaaaatgaccccaaccttttcgttgcactgtatgattttgtggccagtggagataacactctaagcataactaaaggtgaaaagctccgggtcttaggctataatcacaatggggaatggtgtgaagcccaaaccaaaaatggccaaggctgggtcccaagcaactacatcacgccagtcaacagtctggagaaacactcctggtaccatgggcctgtgtcccgcaatgccgctgagtatctgctgagcagcgggatcaatggcagcttcttggtgcgtgagagtgagagcagtcctggccagaggtccatctcgctgagatacgaagggagggtgtaccattacaggatcaacactgcttctgatggcaagctctacgtctcctccgagagccgcttcaacaccctggccgagttggttcatcatcattcaacggtggccgacgggctcatcaccacgctccattatccagccccaaagcgcaacaagcccactgtctatggtgtgtcccccaactacgacaagtgggagatggaacgcacggacatcaccatgaagcacaagctgggcgggggccagtacggggaggtgtacgagggcgtgtggaagaaatacagcctgacggtggccgtgaagaccttgaaggaggacaccatggaggtggaagagttcttgaaagaagctgcagtcatgaaagagatcaaacaccctaacctggtgcagctccttggggtctgcacccgggagcccccgttctatatcatca Ctgagttcatgacctacgggaacctcctggactacctgagggagtgcaaccggcaggaggtgaacgccgtggtgctgctgtacatggccactcagatctcgtcagccatggagtacctggagaagaaaaacttcatccacagagatcttgctgcccgaaactgcctggtaggggagaaccacttggtgaaggtagctgattttggcctgagcaggttgatgacaggggacacctacacagcccatgctggagccaagttccccatcaaatggactgcacccgagagcctggcctacaacaagttctccatcaagtccgacgtctgggcatttggagtattgctttgggaaattgctacctatggcatgtccccttacccgggaattgacctgtcccaggtgtatgagctgctagagaaggactaccgcatggagcgcccagaaggctgcccagagaaggtctatgaactcatgcgagcatgttggcagtggaatccctctgaccggccctcctttgctgaaatccaccaagcctttgaaacaatgttccaggaatccagtatctcagacgaagtggaaaaggagctggggaaacaaggcgtccgtggggctgtgagtaccttgctgcaggccccagagctgcccaccaagacgaggacctccaggagagctgcagagcacagagacaccactgacgtgcctgagatgcctcactccaagggccagggagagagcgatcctctggaccatgagcctgccgtgtctccattgctccctcgaaaagagcgaggtcccccggagggcggcctgaatgaagatgagcgccttctccccaaagacaaaaagaccaacttgttcagcgccttgatcaagaagaagaagaagacagccccaacccctcccaaacgcagcagctccttccgggagatggacggccagccggagcgcagaggggccggcgaggaagagggccgagacatcagcaacggggcactggctttcacccccttggacacagctgacccagccaagtccccaaagcccagcaatggggctggggtccccaatggagccctccgggagtccgggggctcaggcttccggtctccccacctgtggaagaagtccagcacgctgaccagcagccgcctagccaccggcgaggaggagggcggtggcagctccagcaagcgcttcctgcgctcttgctccgcctcctgcgttccccatggggccaaggacacggagtggaggtcagtcacgctgcctcgggacttgcagtccacgggaagacagtttgactcgtccacatttggagggcacaaaagtgagaagccggctctgcctcggaagagggcaggggagaacaggtctgaccaggtgacccgaggcacagtaacgcctccccccaggctggtgaaaaagaatgaggaagctgctgatgaggtcttcaaagacatcatggagtccagcccgggctccagcccgcccaacctgactccaaaacccctccggcggcaggtcaccgtggcccctgcctcgggcctcccccacaaggaagaagctggaaagggcagtgccttagggacccctgctgcagctgagccagtgacccccaccagcaaagcaggctcaggtgcaccagggggcaccagcaagggccccgccgaggagtccagagtgaggaggcacaagcactcctctgagtcgccagggagggacaaggggaaattgtccaggctcaaacctgccccgccgcccccaccagcagcctctgcagggaaggctggaggaaagccctcgcagagcccgagccaggaggcggccggggaggcagtcctgggcgcaaagacaaaagccacgagtctggttgatgctgtgaacagtgacgctgccaagcccagccagccgggagagggcctcaaaaagcccgtgctcccggccactccaaagccacagtccgccaagccgtcggggacccccatcagcccagcccccgttccctccacgttgccatcagcatcctcggccctggcaggggaccagccgtcttccaccgccttcatccctctcatatcaacccgagtgtctcttcggaaaacccgccagcctccagagcggatcgccagcggcgccatcaccaagggcgtggtcctggacagcaccgaggcgctgtgcctcgccatctctaggaactccgagcagatggccagccacagcgcagtgctggaggccggcaaaaacctctacacgttctgcgtgagctatgtggattccatccagcaaatgaggaacaagtttgccttccgagaggccatcaacaaactggagaataatctccgggagcttcagatctgcccggcgacagcaggcagtggtccagcggccactcaggacttcagcaagctcctcagttcggtgaaggaaatcagtgacatagtgcagaggtagcagcagtcaggggtcaggtgtcaggcccgtcggagctgcctgcagcacatgcgggctcgcccatacccgtgacagtggctgacaagggactagtgagtcagcaccttggcccaggagctctgcgccaggcagagctgagggccctgtggagtccagctctactacctacgtttgcaccgcctgccctcccgcaccttcctcctccccgctccgtctctgtcctcgaattttatctgtggagttcctgctccgtggactgcagtcggcatgccaggacccgccagccccgctcccacctagtgccccagactgagctctccaggccaggtgggaacggctgatgtggactgtctttttcatttttttctctctggagcccctcctcccccggctgggcctccttcttccacttctccaagaatggaagcctgaactgaggccttgtgtgtcaggccctctgcctgcactccctggccttgcccgtcgtgtgctgaagacatgtttcaagaaccgcatttcgggaagggcatgcacgggcatgcacacggctggtcactctgccctctgctgctgcccggggtggggtgcactcgccatttcctcacgtgcaggacagctcttgatttgggtggaaaacagggtgctaaagccaaccagcctttgggtcctgggcaggtgggagctgaaaaggatcgaggcatggggcatgtcctttccatctgtccacatccccagagcccagctcttgctctcttgtgacgtgcactgtgaatcctggcaagaaagcttgagtctcaagggtggcaggtcactgtcactgccgacatccctcccccagcagaatggaggcaggggacaagggaggcagtggctagtggggtgaacagctggtgccaaatagccccagactgggcccaggcaggtctgcaagggcccagagtgaaccgtcctttcacacatctgggtgccctgaaagggcccttcccctcccccactcctctaagacaaagtagattcttacaaggccctttcctttggaacaagacagccttcacttttctgagttcttgaagcatttcaaagccctgcctctgtgtagccgccctgagagagaatagagctgccactgggcacctgcgcacaggtgggaggaaagggcctggccagtcctggtcctggctgcactcttgaactgggcgaatgtcttatttaattaccgtgagtgacatagcctcatgttctgtgggggtcatcagggagggttaggaaaaccacaaacggagcccctgaaagcctcacgtatttcacagagcacgcctgccatcttctccccgaggctgccccaggccggagcccagatacgggggctgtgactctgggcagggacccggggtctcctggaccttgacagagcagctaactccgagagcagtgggcaggtggccgcccctgaggcttcacgccgggagaagccaccttcccaccccttcataccgcctcgtgccagcagcctcgcacaggccctagctttacgctcatcacctaaacttgtactttatttttctgatagaaatggtttcctctggatcgttttatgcggttcttacagcacatcacctctttgcccccgacggctgtgacgcagccggagggaggcactagtcaccgacagcggccttgaagacagagcaaagcgcccacccaggtcccccgactgcctgtctccatgaggtactggtcccttccttttgttaacgtgatgtgccactatattttacacgtatctcttggtatgcatcttttatagacgctcttttctaagtggcgtgtgcatagcgtcctgccctgccccctcgggggcctgtggtggctccccctctgcttctcggggtccagtgcattttgtttctgtatatgattctctgtggttttttttgaatccaaatctgtcctctgtagtattttttaaataaatcagtgtttacattagaa

Detection of the ABL T315 mutation is particularly important in patientsexpressing the fusion gene BCR-ABL. In patients with e.g. chronicmyelogenous leukemia (CML), the ABL gene is activated by beingtranslocated within the BCR (breakpoint cluster region) gene onchromosome 22. This fusion gene, BCR-ABL, encodes a tyrosine kinase thatallows cells to proliferate without being regulated by cytokines, whichin turn allows the cell to become cancerous.

As highlighted above, the presence of the ABL T315I mutation in theBCR-ABL fusion protein is significant because patients with thismutation show resistance to tyrosine kinase inhibitors. The substitutioneliminates a critical oxygen molecule needed for hydrogen bondingbetween imatinib and the Abl kinase, and also creates steric hindranceto the binding of most tyrosine kinase inhibitors.

An example BCR-ABL fusion protein (the b2a2 protein) sequence is set outbelow (SEQ ID NO:13). The position of the ABL amino acid correspondingto the ABLT315I mutation is shown in bold and underlined. The amino acidshowing the fusion junction between the BCR and ABL is also shown inbold.

(SEQ ID NO:13)MVDPVGFAEAWKAQFPDSEPPRMELRSVGDIEQELERCKASIRRLEQEVNQERFRMIYLQTLLAKEKKSYDRQRWGFRRAAQAPDGASEPRASASRPQPAPADGADPPPAEEPEARPDGEGSPGKARPGTARRPGAAASGERDDRGPPASVAALRSNFERIRKGHGQPGADAEKPFYVNVEFHHERGLVKVNDKEVSDRISSLGSQAMQMERKKSQHGAGSSVGDASRPPYRGRSSESSCGVDGDYEDAELNPRFLKDNLIDANGGSRPPWPPLEYQPYQSIYVGGMMEGEGKGPLLRSQSTSEQEKRLTWPRRSYSPRSFEDCGGGYTPDCSSNENLTSSEEDFSSGQSSRVSPSPTTYRMFRDKSRSPSQNSQQSFDSSSPPTPQCHKRHRHCPVVVSEATIVGVRKTGQIWPNDGEGAFHGDADGSFGTPPGYGCAADRAEEQRRHQDGLPYIDDSPSSSPHLSSKGRGSRDALVSGALESTKASELDLEKGLEMRKWVLSGILASEETYLSHLEALLLPMKPLKAAATTSQPVLTSQQIETIFFKVPELYEIHKEFYDGLFPRVQQWSHQQRVGDLFQKLASQLGVYRAFVDNYGVAMEMAEKCCQANAQFAEISENLRARSNKDAKDPTTKNSLETLLYKPVDRVTRSTLVLHDLLKHTPASHPDHPLLQDALRISQNFLSSINEEITPRRQSMTVKKGEHRQLLKDSFMVELVEGARKLRHVFLFTDLLLCTKLKKQSGGKTQQYDCKWYIPLTDLSFQMVDELEAVPNIPLVPDEELDALKIKISQIKNDIQREKRANKGSKATERLKKKLSEQESLLLLMSPSMAFRVHSRNGKSYTFLISSDYERAEWRENIREQ

NDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEWCEAQTKNGQGWVPSNYITPVNSLEKHSWYHGPVSRNAAEYLLSSGINGSFLVRESESSPGQRSISLRYEGRVYHYRINTASDGKLYVSSESRFNTLAELVHHHSTVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVKTLKED

SAMEYLEKKNFIHRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAGAKFPIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDLSQVYELLEKDYRMERPEGCPEKVYELMRACWQWNPSDRPSFAEIHQAFETMFQESSISDEVEKELGKQGVRGAVSTLLQAPELPTKTRTSRRAAEHRDTTDVPEMPHSKGQGESDPLDHEPAVSPLLPRKERGPPEGGLNEDERLLPKDKKTNLFSALIKKKKKTAPTPPKRSSSFREMDGQPERRGAGEEEGRDISNGALAFTPLDTADPAKSPKPSNGAGVPNGALRESGGSGFRSPHLWKKSSTLTSSRLATGEEEGGGSSSKRFLRSCSASCVPHGAKDTEWRSVTLPRDLQSTGRQFDSSTFGGHKSEKPALPRKRAGENRSDQVTRGTVTPPPRLVKKNEEAADEVFKDIMESSPGSSPPNLTPKPLRRQVTVAPASGLPHKEEAGKGSALGTPAAAEPVTPTSKAGSGAPGGTSKGPAEESRVRRHKHSSESPGRDKGKLSRLKPAPPPPPAASAGKAGGKPSQSPSQEAAGEAVLGAKTKATSLVDAVNSDAAKPSQPGEGLKKPVLPATPKPQSAKPSGTPISPAPVPSTLPSASSALAGDQPSSTAFIPLISTRVSLRKTRQPPERIASGAITKGVVLDSTEALCLAISRNSEQMASHSAVLEAGKNLYTFCVSYVDSIQQMRNKFAFREAINKLENNLRELQICPATAGSGPAATQDFSKLLSSVKEISDIVQR 

The corresponding mRNA sequence (shown here in cDNA format where base uis replaced by t) is set out below (SEQ ID NO:14), with the position ofthe base that gives rise to the T315I mutation when the base changesfrom c to t is highlighted in bold and underlined and the base showingthe position of the ABL-BCR fusion junction shown in bold).

(SEQ ID NO: 14)ggggggagggtggcggctcgatgggggagccgcctccagggggcccccccgccctgtgcccacggcgcggcccctttaagaggcccgcctggctccgtcatccgcgccgcggccacctccccccggccctccccttcctgcggcgcagagtgcgggccgggcgggagtgcggcgagagccggctggctgagcttagcgtccgaggaggcggcggcggcggcggcggcacggcggcggcggggctgtggggcggtgcggaagcgagaggcgaggagcgcgcgggccgtggccagagtctggcggcggcctggcggagcggagagcagcgcccgcgcctcgccgtgcggaggagccccgcacacaatagcggcgcgcgcagcccgcgcccttccccccggcgcgccccgccccgcgcgccgagcgccccgctccgcctcacctgccaccagggagtgggcgggcattgttcgccgccgccgccgccgcgcgggccatgggggccgcccggcgcccggggccgggctggcgaggcgccgcgccgccgctgagacgggccccgcgcgcagcccggcggcgcaggtaaggccggccgcgccatggtggacccggtgggcttcgcggaggcgtggaaggcgcagttcccggactcagagcccccgcgcatggagctgcgctcagtgggcgacatcgagcaggagctggagcgctgcaaggcctccattcggcgcctggagcaggaggtgaaccaggagcgcttccgcatgatctacctgcagacgttgctggccaaggaaaagaagagctatgaccggcagcgatggggcttccggcgcgcggcgcaggcccccgacggcgcctccgagccccgagcgtccgcgtcgcgcccgcagccagcgcccgccgacggagccgacccgccgcccgccgaggagcccgaggcccggcccgacggcgagggttctccgggtaaggccaggcccgggaccgcccgcaggcccggggcagccgcgtcgggggaacgggacgaccggggaccccccgccagcgtggcggcgctcaggtccaacttcgagcggatccgcaagggccatggccagcccggggcggacgccgagaagcccttctacgtgaacgtcgagtttcaccacgagcgcggcctggtgaaggtcaacgacaaagaggtgtcggaccgcatcagctccctgggcagccaggccatgcagatggagcgcaaaaagtcccagcacggcgcgggctcgagcgtgggggatgcatccaggcccccttaccggggacgctcctcggagagcagctgcggcgtcgacggcgactacgaggacgccgagttgaacccccgcttcctgaaggacaacctgatcgacgccaatggcggtagcaggcccccttggccgcccctggagtaccagccctaccagagcatctacgtcgggggcatgatggaaggggagggcaagggcccgctcctgcgcagccagagcacctctgagcaggagaagcgccttacctggccccgcaggtcctactccccccggagttttgaggattgcggaggcggctataccccggactgcagctccaatgagaacctcacctccagcgaggaggacttctcctctggccagtccagccgcgtgtccccaagccccaccacctaccgcatgttccgggacaaaagccgctctccctcgcagaactcgcaacagtccttcgacagcagcagtccccccacgccgcagtgccataagcggcaccggcactgcccggttgtcgtgtccgaggccaccatcgtgggcgtccgcaagaccgggcagatctggcccaacgatggcgagggcgccttccatggagacgcagatggctcgttcggaacaccacctggatacggctgcgctgcagaccgggcagaggagcagcgccggcaccaagatgggctgccctacattgatgactcgccctcctcatcgccccacctcagcagcaagggcaggggcagccgggatgcgctggtctcgggagccctggagtccactaaagcgagtgagctggacttggaaaagggcttggagatgagaaaatgggtcctgtcgggaatcctggctagcgaggagacttacctgagccacctggaggcactgctgctgcccatgaagcctttgaaagccgctgccaccacctctcagccggtgctgacgagtcagcagatcgagaccatcttcttcaaagtgcctgagctctacgagatccacaaggagttctatgatgggctcttcccccgcgtgcagcagtggagccaccagcagcgggtgggcgacctcttccagaagctggccagccagctgggtgtgtaccgggccttcgtggacaactacggagttgccatggaaatggctgagaagtgctgtcaggccaatgctcagtttgcagaaatctccgagaacctgagagccagaagcaacaaagatgccaaggatccaacgaccaagaactctctggaaactctgctctacaagcctgtggaccgtgtgacgaggagcacgctggtcctccatgacttgctgaagcacactcctgccagccaccctgaccaccccttgctgcaggacgccctccgcatctcacagaacttcctgtccagcatcaatgaggagatcacaccccgacggcagtccatgacggtgaagaagggagagcaccggcagctgctgaaggacagcttcatggtggagctggtggagggggcccgcaagctgcgccacgtcttcctgttcaccgacctgcttctctgcaccaagctcaagaagcagagcggaggcaaaacgcagcagtatgactgcaaatggtacattccgctcacggatctcagcttccagatggtggatgaactggaggcagtgcccaacatccccctggtgcccgatgaggagctggacgctttgaagatcaagatctcccagatcaagaatgacatccagagagagaagagggcgaacaagggcagcaaggctacggagaggctgaagaagaagctgtcggagcaggagtcactgctgctgcttatgtctcccagcatggccttcagggtgcacagccgcaacggcaagagttacacgttcctgatctcctctgactatgagcgtgcagagtggagggagaacatccgggagcagcagaagaagtgtttcagaagcttctccctgacatccgtggagctgcagatgc

agcggccagtagcatctgactttgagcctcagggtctgagtgaagccgctcgttggaactccaaggaaaaccttctcgctggacccagtgaaaatgaccccaaccttttcgttgcactgtatgattttgtggccagtggagataacactctaagcataactaaaggtgaaaagctccgggtcttaggctataatcacaatggggaatggtgtgaagcccaaaccaaaaatggccaaggctgggtcccaagcaactacatcacgccagtcaacagtctggagaaacactcctggtaccatgggcctgtgtcccgcaatgccgctgagtatctgctgagcagcgggatcaatggcagcttcttggtgcgtgagagtgagagcagtcctggccagaggtccatctcgctgagatacgaagggagggtgtaccattacaggatcaacactgcttctgatggcaagctctacgtctcctccgagagccgcttcaacaccctggccgagttggttcatcatcattcaacggtggccgacgggctcatcaccacgctccattatccagccccaaagcgcaacaagcccactgtctatggtgtgtcccccaactacgacaagtgggagatggaacgcacggacatcaccatgaagcacaagctgggcgggggccagtacggggaggtgtacgagggcgtgtggaagaaatacagcctgacggtggccgtgaagaccttgaaggaggacaccatggaggtggaagagttcttgaaagaagctgcagtcatgaaagagatca

tgacctacgggaacctcctggactacctgagggagtgcaaccggcaggaggtgaacgccgtggtgctgctgtacatggccactcagatctcgtcagccatggagtacctggagaagaaaaacttcatccacagagatcttgctgcccgaaactgcctggtaggggagaaccacttggtgaaggtagctgattttggcctgagcaggttgatgacaggggacacctacacagcccatgctggagccaagttccccatcaaatggactgcacccgagagcctggcctacaacaagttctccatcaagtccgacgtctgggcatttggagtattgctttgggaaattgctacctatggcatgtccccttacccgggaattgacctgtcccaggtgtatgagctgctagagaaggactaccgcatggagcgcccagaaggctgcccagagaaggtctatgaactcatgcgagcatgttggcagtggaatccctctgaccggccctcctttgctgaaatccaccaagcctttgaaacaatgttccaggaatccagtatctcagacgaagtggaaaaggagctggggaaacaaggcgtccgtggggctgtgagtaccttgctgcaggccccagagctgcccaccaagacgaggacctccaggagagctgcagagcacagagacaccactgacgtgcctgagatgcctcactccaagggccagggagagagcgatcctctggaccatgagcctgccgtgtctccattgctccctcgaaaagagcgaggtcccccggagggcggcctgaatgaagatgagcgccttctccccaaagacaaaaagaccaacttgttcagcgccttgatcaagaagaagaagaagacagccccaacccctcccaaacgcagcagctccttccgggagatggacggccagccggagcgcagaggggccggcgaggaagagggccgagacatcagcaacggggcactggctttcacccccttggacacagctgacccagccaagtccccaaagcccagcaatggggctggggtccccaatggagccctccgggagtccgggggctcaggcttccggtctccccacctgtggaagaagtccagcacgctgaccagcagccgcctagccaccggcgaggaggagggcggtggcagctccagcaagcgcttcctgcgctcttgctccgcctcctgcgttccccatggggccaaggacacggagtggaggtcagtcacgctgcctcgggacttgcagtccacgggaagacagtttgactcgtccacatttggagggcacaaaagtgagaagccggctctgcctcggaagagggcaggggagaacaggtctgaccaggtgacccgaggcacagtaacgcctccccccaggctggtgaaaaagaatgaggaagctgctgatgaggtcttcaaagacatcatggagtccagcccgggctccagcccgcccaacctgactccaaaacccctccggcggcaggtcaccgtggcccctgcctcgggcctcccccacaaggaagaagctggaaagggcagtgccttagggacccctgctgcagctgagccagtgacccccaccagcaaagcaggctcaggtgcaccagggggcaccagcaagggccccgccgaggagtccagagtgaggaggcacaagcactcctctgagtcgccagggagggacaaggggaaattgtccaggctcaaacctgccccgccgcccccaccagcagcctctgcagggaaggctggaggaaagccctcgcagagcccgagccaggaggcggccggggaggcagtcctgggcgcaaagacaaaagccacgagtctggttgatgctgtgaacagtgacgctgccaagcccagccagccgggagagggcctcaaaaagcccgtgctcccggccactccaaagccacagtccgccaagccgtcggggacccccatcagcccagcccccgttccctccacgttgccatcagcatcctcggccctggcaggggaccagccgtcttccaccgccttcatccctctcatatcaacccgagtgtctcttcggaaaacccgccagcctccagagcggatcgccagcggcgccatcaccaagggcgtggtcctggacagcaccgaggcgctgtgcctcgccatctctaggaactccgagcagatggccagccacagcgcagtgctggaggccggcaaaaacctctacacgttctgcgtgagctatgtggattccatccagcaaatgaggaacaagtttgccttccgagaggccatcaacaaactggagaataatctccgggagcttcagatctgcccggcgacagcaggcagtggtccagcggccactcaggacttcagcaagctcctcagttcggtgaaggaaatcagtgacatagtgcagaggtagcagcagtcaggggtcaggtgtcaggcccgtcggagctgcctgcagcacatgcgggctcgcccatacccgtgacagtggctgacaagggactagtgagtcagcaccttggcccaggagctctgcgccaggcagagctgagggccctgtggagtccagctctactacctacgtttgcaccgcctgccctcccgcaccttcctcctccccgctccgtctctgtcctcgaattttatctgtggagttcctgctccgtggactgcagtcggcatgccaggacccgccagccccgctcccacctagtgccccagactgagctctccaggccaggtgggaacggctgatgtggactgtctttttcatttttttctctctggagcccctcctcccccggctgggcctccttcttccacttctccaagaatggaagcctgaactgaggccttgtgtgtcaggccctctgcctgcactccctggccttgcccgtcgtgtgctgaagacatgtttcaagaaccgcatttcgggaagggcatgcacgggcatgcacacggctggtcactctgccctctgctgctgcccggggtggggtgcactcgccatttcrctcacgtgcaggacagctcttgatttgggtggaaaacagggtgctaaagccaaccagcctttgggtcctgggcaggtgggagctgaaaaggatcgaggcatggggcatgtcctttccatctgtccacatccccagagcccagctcttgctctcttgtgacgtgcactgtgaatcctggcaagaaagcttgagtctcaagggtggcaggtcactgtcactgccgacatccctcccccagcagaatggaggcaggggacaagggaggcagtggctagtggggtgaacagctggtgccaaatagccccagactgggcccaggcaggtctgcaagggcccagagtgaaccgtcctttcacacatctgggtgccctgaaagggcccttcccctcccccactcctctaagacaaagtagattcttacaaggccctttcctttggaacaagacagccttcacttttctgagttcttgaagcatttcaaagccctgcctctgtgtagccgccctgagagagaatagagctgccactgggcacctgcgcacaggtgggaggaaagggcctggccagtcctggtcctggctgcactcttgaactgggcgaatgtcttatttaattaccgtgagtgacatagcctcatgttctgtgggggtcatcagggagggttaggaaaaccacaaacggagcccctgaaagcctcacgtatttcacagagcacgcctgccatcttctccccgaggctgccccaggccggagcccagatacgggggctgtgactctgggcagggacccggggtctcctggaccttgacagagcagctaactccgagagcagtgggcaggtggccgcccctgaggcttcacgccgggagaagccaccttcccaccccttcataccgcctcgtgccagcagcctcgcacaggccctagctttacgctcatcacctaaacttgtactttatttttctgatagaaatggtttcctctggatcgttttatgcggttcttacagcacatcacctctttgcccccgacggctgtgacgcagccggagggaggcactagtcaccgacagcggccttgaagacagagcaaagcgcccacccaggtcccccgactgcctgtctccatgaggtactggtcccttccttttgttaacgtgatgtgccactatattttacacgtatctcttggtatgcatcttttatagacgctcttttctaagtggcgtgtgcatagcgtcctgccctgccccctcgggggcctgtggtggctccccctctgcttctcggggtccagtgcattttgtttctgtatatgattctctgtggttttttttgaatccaaatctgtcctctgtagtattttttaaataaatcagtgtttacattagaa

Accordingly, in a further aspect, the present invention provides amethod of detecting for the presence of a mutation in the ABL genecoding for the ABL T315I mutation, the method comprising:

-   a) providing a sample comprising an mRNA transcript encoding the ABL    T315I mutation;-   b) performing a reverse-transcription reaction to generate a cDNA    sequence from the mRNA transcript; and-   c) amplifying a cDNA sequence generated in step (b);    wherein the reverse-transcription reaction in step (b) is selective    for reverse transcription of the mRNA transcript encoding the ABL    T315I mutation over a corresponding alternative ABL transcript that    does not contain the mutation.

The transcription reaction in this aspect preferably comprises:

-   (i) annealing a reverse primer to a region of the mRNA transcript    comprising a mutation site, wherein the mutation site is responsible    for the ABL T315I mutation; and-   (ii) extending the reverse primer to generate a cDNA sequence from    the mRNA transcript; wherein the mRNA transcript encoding the ABL    T315I mutation and the alternative transcript differ in the identity    of the base at the mutation site, and wherein selectivity for    reverse transcription of the mRNA encoding for the T315I mutation is    achieved, at least in part, by the presence of a base in the reverse    primer which is complementary to the base at the mutant site of the    mRNA but which establishes a mismatch at the base in the    corresponding position of the mutation site in the alternative    transcript. Preferably, the base is at the 3′ end of the reverse    primer.

Step (c) preferably further comprises annealing a forward primer to thecDNA sequence and performing a polymerase chain reaction (PCR) on thecDNA sequence. In a further embodiment, the reverse transcriptionreaction and PCR reaction employ the same reverse primer. The reversetranscription reaction and PCR reaction may be carried out using thesame enzyme, optionally wherein the enzyme is rTth. An example sequencefor the reverse primer is a primer comprising the sequence 5′CCGTAGGTCATGAACTCAA.

It will be appreciated that in this aspect, the ABL T315I mutation maybe present on a fusion protein, particularly a BCR-ABL fusion protein.In such a case, the 315^(th) amino acid in the fusion protein may not bethe same position as the 315^(th) amino acid in non-fused ABL. However,a skilled person would readily be able to identify the correspondingamino acid position in the fusion protein, since a skilled personskilled in the art can readily align similar sequences and locate thesame mutant positions. For example, the amino acid positioncorresponding to the position of the ABL315 mutation site in the fusionprotein sequence recited in SEQ ID NO. 13 is amino acid position 1191.Accordingly, reference herein to the presence of absence of mutant ABLT315I encompasses the detection of the presence or absence of thecorresponding mutant in a fusion protein. This applies where e.g. partof the ABL sequence is truncated so that the amino acid number of themutation position in the fusion protein differs from the amino acidnumber 315. As described above, a skilled person would have nodifficulty in identifying the corresponding position in the fusionprotein by simply taking into account any offset (e.g. truncation) thatmay occur as a result of protein fusion. Where further mutations arerecited elsewhere in the present application, the correspondinginterpretation of the mutations in the context of their presence infusion proteins is to be applied accordingly.

Detection of the ABL mutant can be used to identify how the patient willlikely respond to administration of a drug, such as tyrosine kinaseinhibitor or a BCR-ABL inhibitor e.g. imatinib.

Also provided herein is a method of detecting for the presence of amutation in the ABL gene coding for the ABL T315I mutation, the methodcomprising:

-   a) providing a sample comprising an mRNA transcript;-   b) contacting the sample with reagents capable of performing a    reverse-transcription reaction when mRNA encoding for the ABL T315I    mutation is present, thereby generating a cDNA sequence from the    mRNA transcript when mRNA encoding for the ABL T315I mutation is    present; and-   c) amplifying, if present, a cDNA sequence generated in step (b);    wherein the reverse-transcription reaction in step (b) is selective    for reverse transcription when the mRNA transcript encoding the ABL    T315I mutation is present over a corresponding alternative ABL    transcript that does not contain the mutation.

In a further embodiment of this method, the reagents in step (b)comprise a reverse primer which is selective for reverse transcriptionof the mRNA encoding for the T315I mutation by the presence of a base inthe reverse primer which is complementary to the mRNA base responsiblefor the T315I mutation but which establishes a mis-match in thealternative transcript. The base is preferably at the 3′ end of thereverse primer. The mRNA transcript will typically encodes a BCR-ABLfusion protein or a portion thereof. Where the method of amplificationis PCR amplification, the reverse transcription reaction and PCRreaction may employ the same reverse primer. Also provided herein is amethod of detecting for the absence of a mutation in the ABL gene codingfor the ABL T315I mutation, the method comprising:

-   a) providing a sample comprising an mRNA transcript of ABL coding    for amino acid T at the position corresponding to position 315 of    ABL;-   b) performing a reverse-transcription reaction to generate a cDNA    sequence from the mRNA transcript; and-   c) amplifying a cDNA sequence generated in step (b); wherein the    reverse-transcription reaction in step (b) is selective for reverse    transcription of the mRNA transcript of ABL coding for amino acid T    at the position corresponding to position 315 of ABL over a mutant    transcript coding for amino acid I at the position corresponding to    position 315 of ABL.

In an embodiment of this method, the reverse transcription reactioncomprises:

-   (i) annealing a reverse primer to a region of the mRNA of ABL coding    for amino acid T at the position corresponding to position 315 of    ABL; and-   (ii) extending the reverse primer to generate a cDNA sequence from    the mRNA transcript; wherein selectivity for reverse transcription    of the mRNA is achieved, at least in part, by the presence of a base    which establishes a mis-match in the mRNA transcript encoding the    ABL T315I mutation. The base is preferably at the 3′ end of the    reverse primer. The mRNA transcript will typically encodes a BCR-ABL    fusion protein or a portion thereof. Where the method of    amplification is PCR amplification, the reverse transcription    reaction and PCR reaction may employ the same reverse primer.

Additionally, the invention described herein may provide valuableinformation about the quantity of a mutant transcript present in asample as a means to monitor drug efficacy and disease progression. Forexample, although there is currently no targeted therapy for patientswith a JAK2 V617F mutation there are a number of development effortsunderway which target this mutation in patients with Polycythemia Vera.Monitoring the efficacy of a JAK2 inhibitor with a quantitative JAK2V617F at the transcript level may provide clinically relevantinformation similar to how BCR-ABL RT-PCR is used for monitoring therapyand disease progression in patients with CML.

It will be appreciated that the present invention can be used todetermine or assess any mutant or particular polymorphs is expressed inmRNA. In addition to selecting therapies, or determining diagnosis orprognosis based on mutation or polymorph (e.g. SNP) profile, the presentinvention may also be used to determine the presence (or absence) of anactive gene carrying a mutation by detecting the presence (or absence)of a mutation at the transcript level. This provides an additional levelof confidence that the mutated allele is actually driven from an activepromoter and therefore producing the targeted protein.

Accordingly, the method of the present invention may be used simply todetermine whether the target allele is expressed at the transcriptlevel. Alternatively, or in addition, the present invention may be usedto determine the extent to which the target allele is expressed at thetranscript level.

Whilst the present invention need not be limited to any particulargenes, preferred gene targets include HER2, PI3K, KRAS, EGFR, c-MET,MEK, PTEN, NRAS, HRAS, FGFR1, JAK2, ABL (e.g. ABL T315I), EGFR (e.g.EGFR T790M and/or L858R), MEK, EGFR, BRAF (e.g. BRAF V600E, BRAF V600D,BRAF V600R, BRAF V600K) and ALK. The present invention also provides akit containing reagents for performing the method of the presentinvention. The kit may also contain instructions for performing themethod of the present invention. For example, the kit may containreagents and/or instructions for selectively producing and amplifying acDNA sequence of a target allele of a gene by reverse transcription PCR,wherein the target allele is a mutant allele or is a specific allele ofa polymorphic gene, and wherein the kit comprises:

-   (i) a reverse primer specific to a region of an mRNA transcript of    the target allele comprising a target site, wherein the mRNA    transcript of the target allele of the gene and the mRNA of an    alternative allele of the gene differ in base composition at the    position of the target site, and wherein the reverse primer    comprises one or more bases which are complementary to the mRNA    sequence at the target site of the target allele but which establish    a mis-match at the position of the target site in the alternative    allele;-   (ii) a forward primer specific for an upstream region of the mRNA    transcript of the target allele;-   (iii) a reverse transcriptase; and-   (iv) a DNA polymerase.

In a preferred embodiment of this aspect, the selectivity for reversetranscription of the target allele mRNA over the alternative allele mRNAis achieved, at least in part, by a base at the 3′ end of the reverseprimer which establishes a mis-match with the mRNA sequence of thealternative allele. Preferably, the reverse transcriptase and the DNApolymerase is the same enzyme. In a further preferred embodiment, theenzyme is rTth polymerase. The kit may contain further reagents, forexample, an oligonucleotide probe that allows detection of theamplification product using real-time PCR. Such a probe may contain asequence that hybridises to the amplified cDNA and contains afluorescent dye (the reporter) attached to one end of theoligonucleotide and a quencher, which absorbs light emitted from thereporter when in close proximity to it, is bound to the other end. Thekit may also comprise deoxynucleoside triphosphates (dNTPs).

In a further embodiment, the present invention provides a kit fordetecting the presence of a mutation in the ABL gene coding for the ABLT315I mutation, wherein the kit comprises: (i) a reverse primer specificto a region of an mRNA transcript encoding the ABL T315I mutation, andwherein the reverse primer comprises a base which is complementary tothe mRNA base responsible for the T315I mutation but which establishes amismatch in the corresponding base of the wild type mRNA lacking themutation; (ii) a forward primer specific for an upstream region of themRNA transcript; (iii) a reverse transcriptase; and (iv) a DNApolymerase.

The present invention will now be described with reference to thefollowing non-limiting examples.

EXAMPLES Materials and Methods Cell Lines

Equal amounts of RKO cells were distributed into ten different 1.7 mlsnap top tubes. Total RNA was isolated from five of the tubes using theStratagene RNA Isolation kit and DNA was isolated from the second set offive using the Qiagen DNA Mini kit. The nucleic acid yields wereassessed using a NanoDrop 1000. The concentrations were not normalizedbetween the various samples in an attempt to maintain cell equivalencybetween each tube.

Tissue Sections

Formalin-fixed paraffin embedded tissue sections with known mutationalstatus were cut at 5 uM and mounted on glass slides. H&Es were preparedfrom one section to verify tumor content and the others were used forDNA and RNA isolation. The whole sections were removed from the slidesand incubated in digestion buffer to release the cellular content. DNAand RNA were isolated from the cell lysate using a column basedextraction protocol optimized for fixed and embedded tissues. A NanoDrop1000 was used to assess the quantity and quality of the nucleic acids.As with the cell line studies the concentrations were not normalized inan attempt to maintain an equal number of target cells for eachextraction.

BRAF assay

Allele Specific RT-PCR

An allele specific PCR was developed and optimized for the wild-typesequence as well as the V600E mutation of BRAF. The assay was designedto target either the nucleotide of the wild-type sequence or the V600Emutated sequence at the terminal 3′ nucleotide of the reverse primer.Both the wild-type and mutation specific primer sets were identical withthe exception of the terminal 3′ position of the reverse primer. Aseries of primers were evaluated for analytical sensitivity as wellamplification specificity for each sequence. The primers with theoptimal performance were selected for further studies. The primersequences are identified in below.

The DNA-based PCR was performed in 10 ul volumes using AppliedBiosystems Fast Advanced master mix. The master mix contains allcomponents required to perform PCR in a predesigned formula with theexception of the assay specific primers. The same primer/probe set wasfound to be optimal for both the DNA and mRNA based reactions and wasadded to the reaction at 0.4 uM for each primer and 0.2 uM for theprobe. The probe was an MGB probe labelled with a FAM reporter. Two ulof the DNA preparations were subjected to 40 amplification cycles usingan Applied Biosystems 7900 HT. The cycling parameters were 50° C. for 2minutes, 95° C. for 20 seconds, followed by 40 cycles of 95° C. for 1second and 60° C. for 20 seconds.

The RNA based reactions were also performed in 10 ul but used EZ OneStep Chemistry from Applied Biosystems. The EZ One Step Chemistry hasall components required to perform one-step RT-PCR with the exception ofthe gene specific primers and probe. As previously mentioned theprimer/probe set used for the RT-PCR was the same as was used in the DNAbased assay. The optimal primer/probe concentration for the RT-PCR wasalso 0.4 uM for each primer and 0.2 uM for the probe. As with the DNAbased assay 2 ul of the RNA preparation were subjected to an initialhold 60° C. for 30 minutes for the conversion to cDNA followed, followedby 40 cycles of 95° C. for 15 second and 60° C. for 60 seconds.

BRAF Gene segment (SEQ ID NO: 1) [SEQ ID NO: 1]5′TAATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGG TCTAGCTACAG TGAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGT TGTCTGGATCCATTTTGTGGATGGCACCAGAA

In SEQ ID NO:1, the exon boundaries of exon 15 are defined by thenucleotides at positions 4 and 121. In the V600E mutant the T base atposition 60 of SEQ ID NO: 1 is replaced by A (shown by the underscore).

Reverse primer for wild-type (BRAF RPWT) (SEQ ID NO: 2)5′CAC TCC ATC GAG ATT TC A Tm: 48.2% GC: 44 18 bpReverse Primer for mutant (BRAF RPMUT)) (SEQ ID NO: 3)5′C CAC TCC ATC GAG ATT TC T Tm: 51.1% GC: 47 19 bpForward Primer (BRAF FP, in same exon) (SEQ ID NO: 4)5′ATA TTT CTT CAT GAA GAC CTC ACA GTA Tm: 55.7% GC:33 27 bpReal-time PCT Probe (BRAF Probe) (SEQ ID NO: 5)5′AGG TGA TTT TGG TCT AGC TAC Tm: 70.0% GC: 43 21 bp

The FP/RPWT (Wild-type) primer combination generates a 72 bp product

The FP/RPMUT (Mutation) primer combination generates a 73 bp longproduct.

The FP/RPWT primer combination is used as an amplification control toverify the presence of amplifiable BRAF sequence in the sample.

Results

The BRAF V600E One-Step Allele Specific RT-PCR was capable of detectingthe mutant transcript in each sample that was tested with a knownmutation. FIG. 1 a is an amplification plot generated from a DNA basedAS-PCR while FIG. 1 b demonstrates an amplification plot generated fromthe RNA fraction of the same sample using the AS-RT-PCR methodology.Both fractions were isolated from the same number of cells collectedfrom a tumour biopsy known to harbour the BRAF V600E mutation. FIGS. 1 aand 1 b show amplification plots with Ct values of 31.8 and 25.8,respectively. The difference in Ct values generated between the twomethodologies (delta Ct) is approximately 6 cycles for this sample. Adifference of 6 cycles translates to a 64 fold increase in sensitivityachieved with the RNA based assay over the traditional DNA based AS-PCR.

FIGS. 2 a and 2 b show results generated from the same sample where theAS-RT-PCR provided a more modest advantage over the DNA based assay. TheCt values generated from the DNA and RNA based assays were 24.4 and23.6, respectively. The delta Ct for this sample was 0.8 Ct whichcalculates to about a 1.7 fold increase in sensitivity. Although thedelta Ct value was low, the RNA based assay still provided a sensitivityadvantage over the DNA based approach.

FIGS. 3 a and 3 b also demonstrate variable sensitivities between theDNA and RNA based methodologies. In this sample the DNA based assaygenerated a Ct of 33.2 and the RNA based approach crossed the thresholdat 25.7. The delta Ct for this sample is 7.5 which translates to a 181fold difference in sensitivity between the two methods.

FIGS. 4 a and 4 b demonstrate a sample that likely had an equivalentnumber of mutated DNA copies and mRNA transcripts per cell. The Ctvalues generated from each fraction are almost identical indicating aclose to equal number of targets per fraction. The DNA based assay had aCt of 25.4 while the RNA AS-RT-PCR generated a Ct of 25.3.

FIGS. 5 a and 5 b were generated from a sample that showed a significantenhancement in sensitivity from the RNA based assay. The Ct value fromthe traditional AS-PCR is 34 while the AS-RT-PCR is 26.3. The delta Ctfor this sample is 7.7 cycles between each fraction which is a 208 foldincrease in sensitivity from the AS-RT-PCR methodology. Although somesamples generated similar Ct vales for the DNA and RNA fractions, on nooccasion was a higher Ct value observed for the mRNA fraction whencompared to results generated using DNA for any sample set that wastested. Additionally we were able to generate results from samples thatwere either QNS for DNA based assays or failed to generate a result whentested.

ABL Assay

Allele specific PCR was developed for the T315I mutation. The assay wasdeveloped and optimized for the wild-type reaction as well as themutation. The T315I mutation is a result of a C>T point mutation at the947 nucleotide of the ABL gene. The reverse primers of the assay hadeither a C or T at the last base at the 3′ position to selectivelyamplify the wild-type or mutated sequence. Primers (tablet) werescreened for analytic sensitivity, reaction efficiency and amplificationspecificity using a RNA extracted from a cell line homozygous for theT315I mutation and K562 RNA (Promega). The reactions were performed onan ABI 7900HT using EZ RT-PCR Core Reagents (Applied Biosystems) whichcontained all the necessary components with the exception of the genespecific primers and probe. Two ul of the RNA fraction were subjected toa 60° C. hold for 30 minutes, 95° C. for 15 seconds, followed by 40cycles of 95° C. for 15 second and 60° C. for 60 seconds. EZ One Stepchemistry was chosen to take advantage of the higher reversetranscription temperature associated with rTth polymerase.

TABLE 1 T315I Primers and Probe Name Direction Sequence T315I F Sense 5′CTGGTGCAGCTCCTTGGG (SEQ ID NO: 6) T315I RWT Antisense 5′CCGTAGGTCATGAACTCAG (SEQ ID NO: 7) T315I RMut Antisense 5′CCGTAGGTCATGAACTCAA (SEQ ID NO: 8) T315I Probe Sense FAM-5′CCCCGTTCTATATCATC (SEQ ID NO: 9) *Underscored base represents nucleotidetargeted in each reaction.

While the particular embodiment of the present invention has been shownand described, it will be obvious to those skilled in the art thatchanges and modifications may be made without departing from theteachings of the invention. The matter set forth in the foregoingdescription and accompanying drawings is offered by way of illustrationonly and not as a limitation. The actual scope of the invention isintended to be defined in the following claims when viewed in theirproper perspective based on the prior art.

What is claimed is:
 1. A method of selectively producing and amplifyinga cDNA sequence of a target allele of a gene, wherein the target alleleis a mutant allele or is a specific allele of a polymorphic gene, themethod comprising: a) providing a sample comprising an mRNA transcriptof the target allele; b) performing a reverse-transcription reaction togenerate a cDNA sequence from the mRNA transcript of the target allele;and c) amplifying a cDNA sequence of the target allele generated in step(b); wherein the reverse-transcription reaction in step (b) is selectivefor reverse transcription of the mRNA transcript of the target alleleover an mRNA transcript of an alternative allele of the same gene. 2.The method of claim 1, wherein the target allele is a mutant allele andthe alternative allele is the wild-type allele.
 3. The method of claim1, wherein the target allele is a specific allele of a polymorphic genecomprising a polymorphic site, and the target allele and alternativeallele differ in base composition at the polymorphic site.
 4. The methodof claim 3, wherein the polymorphic site is a single nucleotidepolymorphism (SNP) site.
 5. The method of claim 1, wherein thereverse-transcription reaction comprises: (i) annealing a reverse primerto a region of the mRNA transcript of the target allele comprising atarget site; and (ii) extending the reverse primer to generate a cDNAsequence from the mRNA transcript of the target allele; wherein the mRNAtranscript of the target allele and the mRNA of the alternative allelediffer in base composition at the position of the target site, andwherein selectivity for reverse transcription of the target allele mRNAover the alternative allele mRNA is achieved by the presence of one ormore bases in the reverse primer which are complementary to the mRNAsequence at the target site of the target allele but which establish amismatch at the position of the target site in the alternative allele.6. The method of claim 5, wherein the target site is a mutation site ora SNP site.
 7. The method of claim 5, wherein the reverse primer bindswith full complementarity to the mRNA of the target allele.
 8. Themethod of claim 5, wherein the selectivity for reverse transcription ofthe target allele mRNA over the alternative allele mRNA is achieved, atleast in part, by a base at the 3′ end of the reverse primer whichestablishes a mismatch with the mRNA sequence of the alternative allele.9. The method of claim 5, wherein the reverse primer is between 10 and30 nucleotides in length.
 10. The method of claim 5, wherein step (c)comprises annealing a forward primer to the cDNA sequence and performinga polymerase chain reaction (PCR) on the cDNA sequence.
 11. The methodof claim 10, wherein the reverse transcription reaction and PCR reactionemploy the same reverse primer.
 12. The method of claim 11, wherein theforward primer and reverse primer are specific for the same exon of thetarget allele.
 13. The method of claim 10, wherein the reversetranscription reaction and PCR reaction are carried out using the sameenzyme, optionally wherein the enzyme is rTth.
 14. The method of claim1, wherein the sample is substantially free of DNA.
 15. The method ofclaim 1, wherein the target allele is the mutant allele of the humanBRAF gene encoding a V600 mutation, and the method is selective forproducing and amplifying cDNA of the V600 mutation over cDNA ofwild-type BRAF.
 16. The method of claim 1, further comprising detectingand/or quantifying the presence of the amplified cDNA.
 17. The method ofclaim 16, wherein the amplified cDNA is detected by real-time PCR. 18.The method of claim 1, wherein the target allele is an allele of HER2,PI3K, KRAS, EGFR, c-MET, MEK, PTEN, NRAS, HRAS, FGFR1, JAK2, EGFR, MEK,EGFR or ALK.
 19. The method of claim 1, wherein the target allele isBRAF V600E, BRAF V600D, BRAF V600R, BRAF V600K, EGFR L858R, EGFR T790M,or ALK C1156Y.
 20. The method of claim 1, wherein the presence of thetarget allele is predictive of a diagnosis and/or a prognosis of asubject from which the sample is taken.
 21. The method of claim 20,further comprising detecting the amplified cDNA of the target allele andassessing from the detection of the amplified cDNA a diagnosis and/or aprognosis of the subject.
 22. The method of claim 1, wherein the sampleis from a subject known to have, or suspected to have, a disease, andwherein the presence of the target allele is predictive of how thesubject will respond to administration of a drug to treat the disease.23. The method of claim 22, further comprising detecting the amplifiedcDNA of the target allele and assessing from the detection of theamplified cDNA the likelihood of success of treating the subject withthe drug.
 24. The method of claim 22, wherein the target allele is amutant allele of the human BRAF gene encoding the V600 mutation and thedrug is vemurafenib.
 25. A kit for selectively producing and amplifyinga cDNA sequence of a target allele of a gene by reverse transcriptionPCR, wherein the target allele is a mutant allele or is a specificallele of a polymorphic gene, and wherein the kit comprises: (i) areverse primer specific to a region of an mRNA transcript of the targetallele comprising a target site, wherein the mRNA transcript of thetarget allele of the gene and the mRNA of an alternative allele of thegene differ in base composition at the position of the target site, andwherein the reverse primer comprises one or more bases which arecomplementary to the mRNA sequence at the target site of the targetallele but which establish a mis-match at the position of the targetsite in the alternative allele; (ii) a forward primer specific for anupstream region of the target allele; (iii) a reverse transcriptase; and(iv) a DNA polymerase.
 26. The kit of claim 25, wherein the selectivityfor reverse transcription of the target allele mRNA over the alternativeallele mRNA is achieved, at least in part, by a base at the 3′ end ofthe reverse primer which establishes a mismatch with the mRNA sequenceof the alternative allele.
 27. The kit of claim 25, wherein the reversetranscriptase and the DNA polymerase are the same enzyme.
 28. A methodof detecting for the presence of a gene mutation, the method comprising:a) providing a sample comprising an mRNA transcript; b) contacting thesample with reagents capable of performing a reverse-transcriptionreaction when mRNA containing the mutation is present, therebygenerating a cDNA sequence from the mRNA transcript when mRNA encodingfor the mutation is present; and c) amplifying, if present, a cDNAsequence generated in step (b); wherein the reverse-transcriptionreaction in step (b) is selective for reverse transcription when themRNA transcript containing the mutation is present over the alternativetranscript of the gene that does not contain the mutation.
 29. Themethod of claim 28, wherein the reagents in step (b) comprise a reverseprimer which is selective for reverse transcription of the mRNAcontaining the mutation by the presence of a base in the reverse primerwhich is complementary to the mRNA base containing the mutation butwhich establishes a mismatch in the alternative transcript.
 30. Themethod of claim 29, wherein the base is at the 3′ end of the reverseprimer.
 31. The method of claim 29, where step (c) comprises annealing aforward primer to the cDNA sequence and performing a polymerase chainreaction (PCR) on the cDNA sequence.
 32. The method of claim 31, whereinthe reverse transcription reaction and PCR reaction employ the samereverse primer.
 33. The method of claim 28, wherein the mutation is inHER2, PI3K (PIK3CA), KRAS, EGFR, c-MET, MEK, PTEN, NRAS, HRAS, FGFR1,JAK2, BRAF or ALK.
 34. The method according to claim 1, wherein the mRNAtranscript of the alternative allele is not present in the sample.